Toxins in Renal Disease and Dialysis Therapy: Genotoxic ... · dialysis patients is expected between 2000 and 2015 (Gilbertson, Liu et al. 2005), and 2 million ESRD patients world-wide

Toxins in Renal Disease and Dialysis Therapy:

Genotoxic Potential and Mechanisms

Dissertation zur Erlangung des

naturwissenschaftlichen Doktorgrades der

Bayerischen Julius-Maximilians-Universität Würzburg

vorgelegt von

Kristin Fink

geboren in Magdeburg

Würzburg, 2008

Eingereicht am:………………………………

Mitglieder der Promotionskommission:

Vorsitzender: Herr Prof. Dr. Müller ……………………………….

1. Gutachter: Frau Prof. Dr. Stopper …………..…………………

2. Gutachter: Herr Prof. Dr. Benz ……….………………………..

Tag des Promotionskolloquiums: …………………………………

Doktorurkunde ausgehändigt am: …………………………………

Index

i

Index

Index .........................................................................................................................i

A Introduction.......................................................................................................... 1

1 Kidney and Kidney Disease ............................................................................. 1

1.1 Kidney Anatomy and Function of Kidneys ................................................ 1

1.1.1 Macroscopic Organisation ................................................................. 1

1.1.2 Microscopic Organisation and Function............................................. 2

1.2 Kidney Failure........................................................................................... 2

1.3 Renal Replacement Therapies ................................................................. 3

1.3.1 Hemodialysis ..................................................................................... 3

1.4 Hemodialysers .......................................................................................... 4

1.5 Problems caused by Dialysis .................................................................... 5

1.6 Dialysis Patients and Cancer .................................................................... 5

2 Substances Leaching from Extracorporeal Blood Circuit ................................. 6

2.1 Bisphenol A............................................................................................... 7

2.1.1 Structure and Use.............................................................................. 7

2.1.2 Exposure and Metabolism ................................................................. 7

2.1.3 In vitro and In vivo Effects of BPA ..................................................... 8

2.1.4 Concerns ......................................................................................... 11

2.2 Phthalates............................................................................................... 12

2.2.1 Structure and Use of Di(2-Ethylhexyl)phthalate............................... 12

2.2.2 Exposure to and Metabolism of DEHP ............................................ 13

2.2.3 In vitro and in vivo Effects of DEHP................................................. 15

2.2.4 Concerns ......................................................................................... 18

3 Uremic Toxins ................................................................................................ 19

3.1 Homocysteine ......................................................................................... 20

3.1.1 Chemical Structure and Pathways................................................... 20

3.1.2 Homocysteine Levels....................................................................... 23

3.1.3 Reasons for Elevated Homocysteine Levels ................................... 23

3.1.4 Clinical Implications of Elevated Homocysteine Levels ................... 25

3.1.5 Hyperhomocysteinemia and Cancer................................................ 26

3.1.6 Homocysteine-Thiolactone .............................................................. 27

3.2 Advanced Glycation End-Products (AGEs)............................................. 29

Index

ii

3.2.1 Formation of Advanced Glycation End-Products ............................. 29

3.2.2 Biological Effects of AGEs ............................................................... 30

3.3 Leptin ...................................................................................................... 31

3.3.1 Leptin – an Uremic Toxin?............................................................... 32

4 Cancer ........................................................................................................... 32

4.1 Types of DNA Damage........................................................................... 33

4.1.1 DNA Oxidation................................................................................. 33

4.1.2 Changes in DNA Cytosine-Methylation............................................ 34

B Objectives.......................................................................................................... 35

C Materials & Methods.......................................................................................... 36

1 General Materials........................................................................................... 36

1.1 General Technical Equipment:................................................................ 36

1.2 General Materials and Chemicals........................................................... 37

2 Cell Culture .................................................................................................... 38

2.1 Media, Supplements and General Buffer ................................................ 38

2.2 Cell Lines, Media and Growth Conditions............................................... 39

2.2.1 Maintenance of Cell Culture ............................................................ 39

2.2.2 Passaging of Cells ........................................................................... 40

2.2.3 Thawing of Cells .............................................................................. 40

2.2.4 Freezing of Cells.............................................................................. 41

2.2.5 Treatment of Cells for Testing ......................................................... 41

3 Toxicological Test .......................................................................................... 42

3.1 Frequent Test Substances...................................................................... 42

3.2 Cytotoxicity ............................................................................................. 42

3.2.1 Proliferation ..................................................................................... 42

3.2.2 BrdU Incorporation Assay................................................................ 43

3.3 Genotoxicity ............................................................................................ 45

3.3.1 Comet Assay (Single-Cell Gel Test) ................................................ 45

3.3.2 Micronucleus Test............................................................................ 48

3.3.3 Determination of DNA-Cytosine Methylation by Flow Cytometry..... 51

3.3.4 Determination of DNA-Cytosine Methylation by LC-MS/MS ............ 54

3.4 Oxidative Stress Measurement............................................................... 57

3.4.1 Reactive Oxygen Species Measurement:........................................ 57

3.5 GSH/GSSG – Assay............................................................................... 59

Index

iii

3.6 Apoptosis ................................................................................................ 61

3.6.1 Bisbenzimide Staining: .................................................................... 61

3.6.2 Annexin V Staining and FACS Analysis........................................... 63

3.7 Test for Estrogenic Activity: E-Screen .................................................... 64

3.7.1 Theoretical Background................................................................... 64

3.7.2 Material............................................................................................ 65

3.7.3 Procedure ........................................................................................ 65

3.8 Generation of Advanced Glycation End Products:.................................. 66


4 Extraction of Eluates from Various Dialysers ................................................. 68

4.1 Conditions of Elution............................................................................... 68

4.1.1 Materials: ......................................................................................... 69

4.1.2 Procedure: ....................................................................................... 70

5 HPLC-MS/MS Analysis .................................................................................. 71

5.1 Full Range Scan ..................................................................................... 71


5.1.2 Materials .......................................................................................... 71

5.1.3 Procedure ........................................................................................ 72

5.2 BPA Analysis .......................................................................................... 72


5.2.2 Materials .......................................................................................... 72

5.2.3 Procedure ........................................................................................ 73

5.3 DEHP Analysis ....................................................................................... 73

5.3.1 Theoretical Background:.................................................................. 73

5.3.2 Materials: ......................................................................................... 73

5.3.3 Procedure: ....................................................................................... 74

D Results .............................................................................................................. 75

1 Substances Extracted from Blood Circuits Containing Dialysers and Tubings75

2 HPLC-MS/MS Analysis of Eluates ................................................................. 75

2.1 Total Ion Scan......................................................................................... 75

2.2 Di(2-ethylhexyl)phthalate Analysis.......................................................... 77

2.3 Bisphenol A Analysis .............................................................................. 79

3 Cytotoxicity Testing........................................................................................ 81

3.1 Cell Proliferation ..................................................................................... 82

Index

iv

3.2 Mitosis Frequency................................................................................... 83

3.3 Apoptosis ................................................................................................ 85

4 Genotoxicity Tests ......................................................................................... 86

4.1 Micronucleus Frequency......................................................................... 87

4.2 Comet Assay .......................................................................................... 88

4.3 Test for Estrogenic Activity ..................................................................... 89

4.3.1 E-Screen.......................................................................................... 90

5 Summary of the Toxicity Testing.................................................................... 91

6 Uremic Toxins ................................................................................................ 92

6.1 Homocysteine and Homocysteine-Thiolactone....................................... 92

6.1.1 Cytotoxicity Testing.......................................................................... 92

6.1.2 Genotoxicity Tests ........................................................................... 97

6.1.3 Oxidative Stress............................................................................. 102

6.1.4 GSH............................................................................................... 103

6.1.5 Methylation .................................................................................... 105

6.1.6 BrdU .............................................................................................. 106

6.2 Leptin .................................................................................................... 108

6.2.1 Cytotoxicity Testing........................................................................ 108

6.2.2 Genotoxicity Testing ...................................................................... 108

6.3 Advanced Glycation End Products ....................................................... 110

6.4 Summary of Toxic Effects of Uremic Toxins ......................................... 110

7 Effects of Patient Serum .............................................................................. 111

E Discussion ....................................................................................................... 113

1 Dialysers ...................................................................................................... 113

1.1 BPA....................................................................................................... 113

1.2 DEHP.................................................................................................... 116

2 Toxicity of Eluates........................................................................................ 117

3 Uremic Toxins .............................................................................................. 118

3.1 Homocysteine ....................................................................................... 118

3.1.1 Consequences for the Patient ....................................................... 121

3.2 Homocysteine-Thiolactone ................................................................... 121

3.3 Advanced Glycation End-Products ....................................................... 122

3.4 Leptin .................................................................................................... 122

3.5 Serum of Dialysis Patients .................................................................... 122

Index

v

4 Conclusion ................................................................................................... 123

F Zusammenfassung.......................................................................................... 125

G Summary ......................................................................................................... 129

H Acknowledgements ......................................................................................... 133

I Appendix ......................................................................................................... 134

1 List of Abbreviations..................................................................................... 134

2 Figures......................................................................................................... 137

3 Tables .......................................................................................................... 140

4 References................................................................................................... 141

5 Curriculum vitae ........................................................................................... 154

6 Publications.................................................................................................. 155

7 Ehrenwörtliche Erklärung............................................................................. 156

A Introduction Kidney and Kidney Disease

1

A Introduction

1 Kidney and Kidney Disease

1.1 Kidney Anatomy and Function of Kidneys

1.1.1 Macroscopic Organisation

The kidneys are two bean-shaped organs, which are part of the urinary system

(Fig. A-1). The concave side of the kidney contains an opening – the hilum - which

admits the renal vein, artery, nerves and the ureter. In humans the kidneys are

located on both sides of the spine just below the diaphragm. They are enclosed in a

fibrous renal capsule and embedded in adipose tissue, which absorbs shocks. The

structure of the kidney consists of two parts: (1.) the outer part – the renal cortex –

and (2.) the inner part – the renal medulla. The renal medulla is composed of 10 - 20

renal pyramids. Each pyramid conjoined with the cortex forms a renal lobe. The tip of

each pyramid - the renal papilla – empties into a calyx, which forms the beginning of

the urinary tract.

Fig. A-1 Kidney anatomy (adapted from MedLinePlus, 2007)


2

1.1.2 Microscopic Organisation and Function

The basic functional unit of the kidney is the nephron. More than one million

nephrons per kidney are located within cortex and medulla. They consist of a filtering

component - the renal corpuscle – and a part specialized in reabsorption and

secretion – the renal tubule. The renal corpuscle is composed of the glomerulus and

the Bowman's capsule. The glomerulus is a capillary tuft through which blood flows

under pressure. The pressure forces water and small solutes of the blood to be

filtered through the capillary walls into the Bowman's capsule, thereby forming the

nephric filtrate. The nephric filtrate flows into the renal tubule, which consists of

several sections: the proximal tubule, the loop of Henle and the distal convoluted

tubule. In these sections organic solutes like glucose and amino acids, most of the

water and salts are reabsorbed, while other substances like hydrogen or ammonium

are excreted actively. Finally, urine flows through the collecting duct system, is

drained into the bladder via the ureter and finally excreted.

By producing urine the kidneys fulfil their main functions: excreting metabolic

waste products and maintaining the homeostasis of the organism. While keeping the

homeostasis, the kidneys also regulate the acid-base balance, the blood pressure

and the plasma volume.

1.2 Kidney Failure

Generally, humans can live with reduced kidney function or even with a single

kidney. However, several diseases can threaten the health of a person because they

result in a dramatically diminished kidney function. On one hand there is acute renal

failure, which develops within hours or days and is generally reversible. Reasons for

acute renal failure are e.g. infections, hypotension, medication or kidney stones. On

the other hand there is the slowly progressing disease of chronic renal failure (CRF).

The leading cause for CRF in the western world is diabetes mellitus (US Renal Data

System 2004), followed by high blood pressure and glomerolunephritis, while in third

world countries HIV infection also plays a important role (Lu and Ross 2005).

After years of suffering from CRF, the glomerula filtration rate of the kidney finally

drops below 15%, leading to end-stage renal disease (ESRD). When ESRD is

reached, renal replacement therapy, like renal transplantation or hemodialysis is

necessary. In the beginning of 2006 more than 64,000 patients in Germany

depended on hemodialysis. Due to demographic changes, increased prevalence of


3

diabetes and higher life expectancy of ESRD patients, the number of dialysis patients

increases about 4.8% per year and will reach 100,000 within the next few years (Frei

and Schober-Halstenberg 2006).

Of course, this problem is not limited to Germany. For the USA a 32% increase of

dialysis patients is expected between 2000 and 2015 (Gilbertson, Liu et al. 2005),

and 2 million ESRD patients world-wide are expected by 2010 (Lynsaght 2002).

Due to the lack of donor kidneys most patients will have to be treated by

hemodialysis.

1.3 Renal Replacement Therapies

The method of artificial kidney replacement is called dialysis: This method does

not heal the underling kidney disease, but it allows removal of waste products – the

so called uremic toxins (see page 19) - and excess fluid from the blood of the patient.

Two dialysis methods are currently available: peritoneal dialysis and hemodialysis

(HD). HD is the most frequent renal replacement therapy with about 180 million

applications worldwide per year.

1.3.1 Hemodialysis

In HD, the arterial blood is pumped from the fore-arm vein of a patient through

tubes into the blood compartment of a dialyser, where it flows through ca. 10,000

hollow fibers with walls of semipermeable membranes. On the other side of the

membranes, a dialysis solution is pumped in counter current flow through the

dialysate compartment of the dialyser (Fig. A-2), allowing the diffusion of waste

products from the blood-compartment into the dialysis fluid compartment. In order to

enhance the natural diffusion alongside the concentration gradient, blood is pumped

at 250 - 300 ml/min while the dialysis fluid flows at 500 ml/min. The semipermeable

membrane contains pores large enough to allow water and uremic toxins to pass

across. After flowing through the dialyser the dialysis fluid is discharged while the

cleansed blood is pumped back into the body. In general HD is performed three times

a week for 4 - 5 h.

More efficient techniques for HD are hemodiafiltration and hemofiltration where a

convective flow in the sense of solvent drag is applied. Hemodiafiltration and

hemofiltration are applied preferentially when larger toxin molecules are removed, i.e.

peptides or small proteins with MW of > 10,000 Da.


4

Fig. A-2 Schematic picture of a hemodialysis circuit

1.4 Hemodialysers

Currently a wide spectrum of hemodialysers combined with different membranes

is available (Tab. A-1). Generally, membranes are produced by two families of

polymers: synthetic and cellulosic. These classes of membranes can be subdivided

into high-flux membranes (large pores) or low-flux membranes (small pores). High

flux membranes allow higher water flux and better removal of high molecular weight

uremic solutes than low flux membranes (Boure and Vanholder 2004).

The polymer of the membrane determines the physical, chemical and biological

properties of a dialysis membrane. Ideally a membrane is highly biocompatible,

adsorbs dialysate impurities from the dialysis fluid, removes middle molecules and is

resistant to all chemical and sterilizing agents used in HD procedures (Boure and

Vanholder 2004; Uhlenbusch-Körwer, Bonnie-Schorn et al. 2004). Given that

synthetic membranes are superior to cellulosic membranes in most of these

properties - especially in biocompatibility – there is a trend towards synthetic polymer

material (Vienken and Bowry 2002).


5

Celluliosic Synthetic

Unmodified (low-flux)

Modified/

regenerated

(high-flux):

Low-flux High-flux

• Cuprammonium rayon

• Cellulose triacetate

• Polysulfone • Polysulfone

• Cellulose diacetate

• Polycarbonate • Polyamide

• Polyamide and

Polysulfone blends

• Polyethersulfone

• Polyacrylonitrile

• Polymethyl-methacrylate

Tab. A-1 Types of membranes with examples (not exhaustive);

(Boure and Vanholder 2004):

1.5 Problems caused by Dialysis

Unfortunately, HD treatment can also produce side-effects. Common problems

are: (1.) the “first-use syndrome”- an allergic reaction towards materials of medical

devices or residues of sterilisation (Charoenpanich, Pollak et al. 1987), (2.) bacterial

or endotoxin contamination by improper treatment of water, dialysate, dialysis

machines and dialysers (Nicholls and Platts 1985; Gordon, Drachman et al. 1990;

Pegues, Beck-Sague et al. 1992; Burwen, Olsen et al. 1995) and (3) contamination

by leachable degradation products of dialyser membranes (Lucas, Kalson et al.

2000).

1.6 Dialysis Patients and Cancer

On top of these problems dialysis patients are at increased risk of cancer,

especially cancer of the urinary tract (Maisonneuve, Agodoa et al. 1999; Teschner,

Garte et al. 2002; Stewart, Buccianti et al. 2003; Vajdic, McDonald et al. 2006) (Tab.

A-2). The risk of kidney cancer rises significantly with time on dialysis (Stewart,

Buccianti et al. 2003). The risk is also higher in young than in old patients and higher

in females compared to males (Stewart, Buccianti et al. 2003).

A Introduction Substances Leaching from Extracorporeal Blood Circuit

6

SIR (95% confidence interval)

Site Australia and New Zealand

Europe USA

All but skin 1.8 (1.7 - 2.0) 1.1 (1.0 - 1.1) 1.2 (1.2 - 1.2)

Oral cavity 1.4 (0.9 - 2.4) 0.6 (0.5 - 0.7) 1.3 (1.2 - 1.4)

Respiratory 1.5 (1.1 - 1.9) 0.9 (0.9 - 1.0) 1.1 (1.1 - 1.2)

Bone, skin, breast 1.4 (1.1 - 1.8) 1.0 (0.9 - 1.1) 0.8 (0.8 - 0.9)

Hemopoietic 1.6 (1.1 - 2.3) 1.3 (1.2 - 1.4) 2.5 (2.4 - 2.6)

Digestive 1.2 (1.0 - 1.5) 0.9 (0.9 - 1.0) 1.2 (1.2 - 1.3)

Genitourinary

All 3.0 (2.6 - 3.5) 1.4 (1.4 - 1.4) 1.1 (1.1 - 1.1)

Bladder 4.8 (3.6 - 6.2) 1.5 (1.4 - 1.7) 1.4 (1.3 - 1.5)

Kidney 9.9 (7.7 - 12.3) 3.3 (3.1 - 3.6) 3.7 (3.5 - 3.9)

Other and unspecific

All 2.3 (1.7 - 3.1) 1.1 (1.0 - 1.2) 2.2 (2.0 - 2.4)

Thyroid 5.9 (3.3 - 10.7) 1.9 (1.5 - 2.3) 2.4 (2.1 - 2.8)

Tab. A-2 Site-specific cancer risk in ESRD patients (Maisonneuve, Agodoa et al. 1999)

Several factors may contribute to the increased cancer incidence: chronic

infections, a weakened immune system, pre-treatment with immunosuppressive

drugs, nutritional deficiencies or the depressed DNA repair in CRF patients (Malachi,

Zevin et al. 1993; Maisonneuve, Agodoa et al. 1999).

Other possible factors which could contribute are: (1) the accumulation of

genotoxic uremic toxins in the blood of the patients or (2) substances leaching from

extracorporal blood circuit into the blood of HD patients (e.g. bisphenol A or di(2-

ethylhexyl) phthalate).

2 Substances Leaching from Extracorporeal Blood

Circuit

During HD blood can be exposed to a variety of compounds derived from tubing,

membranes, dialysis fluid and housing. Especially disconcerting are substances with

known toxic properties or endocrine disrupting chemicals. The term endocrine

disruptor is commonly used to describe environmental agents which alter the

endocrine system, by interacting with hormone receptors (Reviewed by (McLachlan

2001)). Thereby, they can cause alterations in the hormone level leading to infertility,

feminisation of males, reproductive tract malformation, endometriosis and tumours in

estrogen-responsive tissues (McLachlan 2001; Wozniak, Bulayeva et al. 2005).


7

Those effects have preferentially been reported in animal models, e.g. the mouse

model.

Two substances possessing those properties and known to leach from

extracorporal blood circuits have raised special concern: di(2-ethylhexyl)phthalate

(DEHP) and bisphenol A (BPA).

2.1 Bisphenol A

2.1.1 Structure and Use

BPA is the common name for 2,2-(4,4-dihydroxy-diphenyl)propane (Fig. A-3). It is

synthesized by condensation of two equivalents phenol with one equivalent acetone

at low pH and high temperatures.

Fig. A-3 Molecular structure of bisphenol A

The main application of BPA is as monomer component for polycarbonate plastic

and epoxy resins (Staples, Dorn et al. 1998). Consequently, it is used in numerous

consumer products (e.g. food packaging) and dentistry (e.g. brackets, dental fillings).

Unreacted BPA residues, or BPA resulting from hydrolysis of the ester bonds, can

leach from plastics into food, water (Imai and Komabayashi 2000; Bae, Jeong et al.

2002; Lopez-Cervantes and Paseiro-Losada 2003; Sajiki and Yonekubo 2003; Sajiki

and Yonekubo 2004), or saliva and is ingested (Suzuki, Ishikawa et al. 2000;

Watanabe, Hase et al. 2001; Atkinson, Diamond et al. 2002; Watanabe 2004).

2.1.2 Exposure and Metabolism

While the major route of exposure is by food (Scientific committee on toxicology

2002), HD patients obtain an additional burden of BPA which leaches off the dialyser

directly into the blood (Haishima, Hayashi et al. 2001; Yamasaki, Nagake et al. 2001;

Murakami, Ohashi et al. 2007). This is important to notice because orally absorbed


8

BPA undergoes an extensive first-pass effect in the liver, resulting in the detoxified

form of BPA: BPA-glucuronide. When BPA leaches directly into the blood (or is

applied i.p. in test animals) more of the highly bioavailable, free BPA circulates and

stronger effects can be the result (Scientific committee on toxicology 2002).

Fortunately, BPA has no tendency to accumulate and a half-life of less than one day

was described (Pottenger, Domoradzki et al. 2000; Takahashi and Oishi 2000).

Following oral exposure BPA is rapidly absorbed from the gastrointestinal tract in

rodents. However, it is not possible to quantify the actual extent of absorption

because the major route of excretion is via the faeces as parent BPA (50 - 80%

depending on species, strain and gender). The parent BPA can result from two

different sources: It is either 1.) BPA which passes the intestinal tract unchanged and

is not absorbed or 2.) its glucuronide form which is transported into the intestine via

bile and is hydrolysed later on. The excretion route of secondary importance is via

the urine in the form of BPA-glucuronide. Ten additional metabolites could be

detected in urine of mice but nearly no unmodified BPA (Pottenger, Domoradzki et al.

2000; Snyder, Maness et al. 2000; Elsby, Maggs et al. 2001; EU-Report 2003; Zalko,

Soto et al. 2003).

Nevertheless, parent BPA could be detected in the serum of pregnant females by

derivatisation - GC/MS ranging from 0.3 to 18.9 ng/ml (Schönfelder, Wittloht et al.

2002). BPA could even be detected in foetal plasma (0.2 - 9.2 ng/ml). Several other

studies detected between 0.32 and 2.59 ± 5.23 ng/ml BPA in plasma, though mostly

by less sensitive detection methods (for review see (Welshons, Nagel et al. 2006)).

These values are in line with BPA levels in urine: 0.04 µg/l – 8 µg/l (Calafat,

Kuklenyik et al. 2005).

2.1.3 In vitro and In vivo Effects of BPA

2.1.3.1 Acute Toxicity

The acute toxicity test for BPA determined oral LD50 values above 2,000 mg/kg

bw for laboratory animals (NTP 1982). Since the BPA levels in nature and humans

are far lower, it can be concluded that the acute toxicity is not of concern for humans.

Therefore research focus has focussed on possible carcinogenic/mutagenic and

estrogenic effects.


9

2.1.3.2 Reproductive and Developmental Toxicity

Until recently BPA has generally been considered a relatively weak estrogen.

Depending on the test system, the binding affinity of BPA to estrogen receptor α (ER-

α) or β is 2 - 4 orders of magnitude lower than that of 17β estradiol (Feldman and

Krishnan 1995; Dodge, Glasebrook et al. 1996; Kuiper, Lemmen et al. 1998;

Maruyama, Fujimoto et al. 1999). The glucuronide shows even less estrogenic

activity (Snyder, Maness et al. 2000; Matthews, Twomey et al. 2001). However, a

recent study showed that BPA not only acts via genomic responses of the ER, but

also through non-genomic membrane-initiated pathways (Wozniak, Bulayeva et al.

2005). Nanomolar concentrations of BPA resulted in an increased Ca2+ influx in vitro.

This may lead to changes in the signalling process or hormone secretion.

In vivo BPA is 10,000 fold less potent in inducing uterotrophic effects in

ovariectomized rats than 17β-estradiol (Milligan, Balasubramanian et al. 1998). An

uterotropic effect in rats was confirmed after high oral or subcutaneous dosing by

several other groups (Ashby and Tinwell 1998; Laws, Carey et al. 2000; Matthews,

Twomey et al. 2001; Ashby and Odum 2004).

To elucidate the relevance of these observations regarding reproductive toxicity,

an elaborate three generation study on CD Sprague-Dawley rats was conducted (Tyl,

Myers et al. 2002). BPA dosage of up to 5 mg/kg bw per day did not cause any

effects. Reproductive toxicity could only be observed at concentrations which also

produced systemic toxicity (> 50 mg/kg bw per day). This was in line with an earlier

study, which also concluded that BPA is no selective reproductive toxicant (no effect

up to 640 mg/kg bw per day) (Morrissey, George et al. 1987). Tinwell et al also

detected reduced sperm count and a delay in the day of vaginal opening in Alderlay

Park rats only at high doses (50 mg/kg bw per day) (Tinwell, Haseman et al. 2002).

In contrast to those high-dose studies, several groups report effects already at a

low-dose level in mice. While there is no dispute on the high-dose effects, low dose

effects are less clear, especially because many of the reported effects could not be

reproduced in large animal trials.

Low oral doses of BPA (2 - 20 µg/kg bw per day) have been reported to affect

male reproductive organs such as preputial glands and epididymides and to reduce

sperm production (Nagel, vom Saal et al. 1997; vom Saal, Timms et al. 1997; vom

Saal, Cooke et al. 1998). However, other groups could not confirm these

observations; even though the same animal strains were used (Ashby, Tinwell et al.


10

1999) and additional doses were tested (Cagen, Waechter et al. 1999; Cagen,

Waechter et al. 1999). Low-dose oral BPA administration during gestation had no

adverse effect on female offspring in regard to rat puberty development and

reproductive functions in CF1 mice (20 µg/kg/day) (Ashby and Tinwell 1998), or

female and male SD rats (3 mg/kg bw per day) (Nagao, Saito et al. 1999). In utero

exposure of SD rats and Alderlay Park rats (20 µg/kg bw – 100 µg/kg bw) did not

influence litter size, weight, anogenital distance at birth, first estrus, days of vaginal

opening or weight of reproductive organs.

Even though the discussion about the low-dose effect is still ongoing it might be

of relevance for humans: a small preliminary study on Japanese women (n = 77)

found a correlation between plasma BPA level and subsequent miscarriages

(Sugiura-Ogasawara, Ozaki et al. 2005).

2.1.3.3 Mutagenicity

Another concern about constant low-dose BPA exposure is the possible

carcinogenic mutagenic capacity of BPA. Two studies detected DNA adduct

formation following BPA incubation with purified DNA (Atkinson and Roy 1995;

Atkinson and Roy 1995). However, the adduct formation was distinctly decreased in

the presence of inhibitors of cytochrom P450. Therefore the relevance for the in vivo

situation is uncertain. Additionally BPA inhibited microtubule polymerisation in cell

free systems (Metzler and Pfeiffer 1995; Pfeiffer, Rosenberg et al. 1997)

In contrast to the results of cell-free systems, BPA was not mutagenic in the

Ames test of variety of Salmonella typhimurium strains, with and without metabolic

activation (Andersen, Kiel et al. 1978; Haworth, Lawlor et al. 1983; Tennant,

Stasiewicz et al. 1986; Schweikl, Schmalz et al. 1998)

Studies in mammalian cells yielded mixed results. BPA was not mutagenic in

mutation tests with mouse lymphoma L5178Y cells (Myhr and Caspary 1991),

Chinese hamster V79 cells (Schweikl, Schmalz et al. 1998) and Syrian hamster

embryo (SHE) cells (Tsutsui, Tamura et al. 1998).

However, it produced positive results in transformation assays in the same study

(Tsutsui, Tamura et al. 1998). BPA (µM) caused aneuploidy in somatic cells (Tsutsui,

Tamura et al. 2000) and induced micronuclei (MN) in V79 cells (Pfeiffer, Rosenberg

et al. 1997). Furthermore, 100 - 200 µM BPA caused aberrant spindle formation in

V79 cells (Ochi 1999) and in vivo in mice oocytes at environmentally relevant doses


11

(Hunt, Koehler et al. 2003; Susiarjo, Hassold et al. 2007). Even nanomolar

concentrations had the effect of inducing proliferation of the human prostate cancer

cell line LNCaP (Wetherill, Petre et al. 2002).

2.1.3.4 Carcinogenicity

A well conducted two-year carcinogenicity study of the US National Toxicology

Program concluded that there is no convincing evidence for carcinogenic potential of

BPA in B6C3F1 mice (up to 5,000 ppm BPA for male mice approximately 833 mg/kg

bw per day; up to 10,000 ppm BPA for female mice approximately 1666 mg/kg bw

per day.) and F344 rats (up to 2,000 ppm BPA; approx. 100 mg/kg bw per day) (NTP

1982). However, there was a marginal but statistically significant increase in

leukaemia in male rats, along with a not statistically significant increase in leukaemia

in female rats. The male mice showed a marginal significant increase of lymphomas

and leukemias.

Another study also found a marginal increase of leukaemia in F344 rats (2,000

ppm BPA approximately 100 mg/kg bw) and lymphoma, as well as leukaemia in low-

dose (5,000 ppm BPA approximately 833 mg/kg bw) male B6C3F1 mice (Huff 2001).

Therefore the authors concluded that an association of BPA exposure with increased

cancers of the hematopoietic system cannot be ruled out.

Carcinogenesis studies on humans cannot be performed, but epidemiological

studies on humans found no correlation between breast cancer incidence and BPA

exposure in American women (Aschengrau, Coogan et al. 1998).

2.1.4 Concerns

With regard to these results the European Chemicals Bureau concluded that,

although there is need for additional studies, BPA poses no risk for consumers (EU-

Report 2003). The tolerable daily intake (TDI) was even raised from 0.01 mg/kg bw

per day to 0.05 mg/kg bw per day by the EFSA panel (European Food Safety

Authority) (EFSA 2006). BPA levels which normally absorbed are in the low µg/kg

bw range. Therefore they are below the TDI range and the potential hazard for

humans was regarded as minimal (EU-Report 2003). Solely workers handling BPA

may exceed the TDI and may therefore be at risk. Another evaluation by Haighton et

al. following the weight-of-evidence approach as advised by the Internation Agency

for Research on Cancer (IARC) and US Environmental Protection Agency (US EPA)

concluded that BPA is not likely to be carcinogenic to humans (Haighton, Hlywka et


12

al. 2002). Additional evaluation by the Scientific Committee on Toxicology,

Ecotoxicity and the Environment (CSTEE) followed this argumentation (Scientific

committee on toxicology 2002). However, this conclusion is challenged by other

groups (Huff 2001; vom Saal and Hughes 2005).

As the debate is ongoing at the moment, the importance of additional information,

especially regarding special risk populations like HD patients is obvious.

2.2 Phthalates

Phthalates or phthalate esters are a group of chemicals which share a common

chemical structure: they are dialkyl or alkyl/alcaryl esters of 1, 2-benzenedicaroxylic

acid. In Western Europe about one million tonnes of phthalates are produced

annually. Most of it is used as plasticizers to impart flexibility to plastics

(Intermediates 2007). One of the most widely used phthalates is di(2-ethylhexyl)

phthalate (DEHP).

2.2.1 Structure and Use of Di(2-Ethylhexyl)phthalate

DEHP is also known as bis(2-ethylhexyl) phthalate (BEHP) or dioctyl phthalate

(DOP) (Fig. A-4).

O

O

O

O

CH2

CH

CH2

CH3

(CH2)3

CH3

CH2

CH (CH2)3

CH3

Fig. A-4 Molecular structure of di-(2-ethylhexyl)phthalate

DEHP is synthesized by esterification of phthalic acid anhydride with 2-

ethylhexanol.

Its primary use is as plasticizer in PVC, e.g. in flooring and food storage

containers, but it is also used in paints, lubricants and clothing. Another important

application is in medical care products like blood bags, transfusion bags and tubings,

catheters or air tubes (Calafat and McKee 2006; Umweltbundesamt 2006).


13

PVC can contain up to 40% DEHP. DEHP is not covalently bound to the plastic

and can therefore leach or migrate from it. It is easily absorbed by aliphatic

substances.

2.2.2 Exposure to and Metabolism of DEHP

The major route of exposure is via ingestion or inhalation (ATSDR 2002; Barrett

2006). However, patients undergoing certain medical treatments like intubation, i.v.

nutrition, blood transfusion or HD are especially exposed to DEHP. Due to its

lipophilic nature DEHP leaches into the blood easily.

Only a very limited amount of toxicokinetic studies in humans is available; most

of the studies are performed in rodents, following oral exposure. After oral exposure

of up to 200 mg/kg bw DEHP around 50% of the dose is absorbed in non-human

primates (marmosets). At higher concentrations the absorption seems to be dose

limited (Rhodes, Elcombe et al. 1983). If humans are exposed to DEHP iv, 100% is

bioavailable. After absorption DEHP is rapidly metabolised in the intestine and liver to

its corresponding monoester - mono(2-ethylhexyl)phthalate (MEHP) - and 2-

ethylhexanol (Albro and Thomas 1973). Due to its weak polarity MEHP cannot be

excreted directly. It undergoes oxidations by which secondary products like mono-[2-

ethyl-5-hydroxylhexyl] phthalate (5OH-MEHP), mono-[2-ethyl-5-oxylhexyl]phthalate

(5oxo-MEHP), mono-[2-ethyl-5-carboxypentyl]phthalate (5cx-MEPP), mono-[2-

(carboxymethyl)hexyl]phthalate (5cx-MMHP) are formed (Fig. A-5). Both, the

monoester and the oxidative metabolites can be conjugated to glucuronic acid and

excreted in urine or faeces. (ATSDR 2002; Koch, Bolt et al. 2004; Koch, Bolt et al.

2005; Calafat and McKee 2006; Koch, Preuss et al. 2006). Depending on the study

and species 4 – 68% (humans: 15-25%) of the DEHP is excreted by urine with a half

life of 6 - 18 h (Ikeda, Sapienza et al. 1980; Peck and Albro 1982; Schmid and

Schlatter 1985; Astill, Barber et al. 1986).


14

Fig. A-5 Metabolism of di(2-ethylhexyl)phthalate (adapted after Koch, Preuss et al. 2006)

In order to estimate the DEHP burden of the general population, urinary

metabolites of DEHP – mostly MEHP – have been measured by various groups (Tab.

A-3).

Study n MEHP 5OH-MEHP 5oxo-MEHP

Blount, Silva et al. 2000 289 2.7 n.a. n.a.

Hoppin, Brock et al. 2002 46 7.3 n.a. n.a.

Koch, Rossbach et al. 2003 85 10.3 46.8 36.5

Barr, Silva et al. 2003 62 4.5 35.9 28.3

Kato, Silva et al. 2004 176 < LOD 17.4 15.6

Tab. A-3 Median body burden of DEHP, expressed in the urinary concentration of its

metabolites (in µg/ml)

n.a., not analysed; MEHP, mono(2-ethylhexyl)phthalate; 5OH-MEHP,

mono-[2-ethyl-5-hydroxylhexyl] phthalate; 5oxo-MEHP, mono-[2-ethyl-5-oxylhexyl]

phthalate


15

Based on the metabolites detected in urine, the actual DEHP intake is estimated

to be ≈ 30 µg/kg bw per day (Doull, Cattley et al. 1999) or 5.6 to 21 µg/kg bw per day

in adults and 7.7 to 25 µg/kg/day in children (Koch, Preuss et al. 2006).

Estimations about the additional burden of HD patients range from 3.6 to 150 mg

per dialysis session (Pollack, Buchanan et al. 1985; Flaminio, Bergia et al. 1988;

Faouzi, Dine et al. 1999; Dine, Luyckx et al. 2000).

2.2.3 In vitro and in vivo Effects of DEHP

2.2.3.1 Acute Toxicity

The acute oral toxicity of DEHP is very low. The LD50 in rat is > 20,000 mg/kg

(NTP 1982) and > 9,860 mg/kg in mice (Nuodex 1981).

However, it is very likely that the metabolites, not DEHP itself, are the bioactive

forms (Calafat and McKee 2006). DEHP or its metabolites produce a wide spectrum

of toxic effects in multiple organ systems of laboratory animals like liver, reproductive

tract, kidneys, lungs and heart (reviewed in Tickner, Schettler et al. 2001; Bureau

2003). However, these symptoms did only develop at a very high dosages (> 100

mg/kg bw to several g/kg bw), which are not relevant for the in vivo situation.

However, a lot of concern has been raised about possible toxicity at lower doses

especially reproductive toxicity and mutagenic and hepato-carcinogenic effects in

laboratory animals.

2.2.3.2 Mutagenicity

The possible genotoxic effects of DEHP and its metabolites have been

thoroughly investigated. DEHP and its metabolites were tested negative in Ames

tests of several Salmonella typhimurium strains with and without metabolic activation

(Zeiger, Haworth et al. 1982; Kirby, Pizzarello et al. 1983; Yoshikawa, Tanaka et al.

1983; Zeiger, Haworth et al. 1985; Schmezer, Pool et al. 1988; Dirven, Theuws et al.

1991).

DEHP and its metabolites were also non-mutagenic in mouse lymphoma L5178Y

mutation tests (Kirby, Pizzarello et al. 1983; Astill, Barber et al. 1986), did not induce

DNA damage, chromosomal aberrations or sister chromatide exchange in Chinese

hamster ovary cells (Phillips, James et al. 1982; Douglas, Hugenholtz et al. 1986),

and did not induce DNA damage or repair in mouse, rat or human hepatocytes

(Butterworth, Bermudez et al. 1984; Smith-Oliver and Butterworth 1987). They also


16

tested negative in micronucleus assays (Astill, Barber et al. 1986; Douglas,

Hugenholtz et al. 1986). However, a few studies report induction of cell

transformation in Syrian hamster embryo cells (Sanner, Mikalsen et al. 1991;

Mikalsen and Sanner 1993; Tsutsui, Watanabe et al. 1993) and DNA damage

detectable by comet-assay in human lymphocytes (Anderson, Yu et al. 1999). Most

in vivo studies for DNA damage/repair and DNA adduct formation in DEHP feeding

studies were negative up to 1000 mg/kg bw day (Butterworth, Bermudez et al. 1984;

Kornbrust, Barfknecht et al. 1984; Gupta, Goel et al. 1985; Smith-Oliver and

Butterworth 1987). Only one study reported increased 8-OH-dG levels (2-fold) in rat

liver after one month DEHP containing diet (Takagi, Sai et al. 1990); another study

reported a 5-fold increase in single strand breaks, but only in tumour-bearing rats

(Tamura, Iida et al. 1991). They concluded that this effect is not due to direct

genotoxicity. With regard to these results the US EPA and EU Commission classified

DEHP as not mutagenic (Doull, Cattley et al. 1999; Bureau 2003).

2.2.3.3 Carcinogenicity

Even though DEHP and its metabolites are not genotoxic, several feeding studies

conclude that it is hepato-carcinogenicity in rodents. A long-term feeding study of the

US National Toxicology Program showed that DEHP induced hepatocellular

carcinomas in F344 rats and B6C3F1 mice in a dose-dependent manner (Kluwe,

Haseman et al. 1982; NTP 1982). These results were confirmed by other groups

(Cattley, Conway et al. 1987; Popp, Garvey et al. 1987; Rao, Usuda et al. 1987;

David, Moore et al. 1999). The lowest observed adverse effect level (LOAEL) derived

from those studies for rats is 147 mg/kg bw per day and for mice 292 mg/kg bw per

day DEHP in the diet.

The mechanism through which DEHP induces liver tumours in rodents is

probably by peroxisome proliferation. Peroxisomes are cytoplasmic organelles which

contain a number of hydrogen peroxide generating oxidases, catalases and fatty acid

β-oxidation enzymes (Reddy 2004).

The ability of DEHP to act as peroxisome proliferator is due to its metabolite

MEHP. MEHP interacts with the peroxisome proliferator–activated receptor α (Ppar-

α), thereby increasing the size and the number of peroxisomes in vivo (Moody and

Reddy 1978.; Rao and Reddy 1991; Reddy 2004). Ppar-α activation also leads to

changes in gene expression, e.g. increased β-oxidation and ω-oxidation.


17

Subsequently more H2O2 is generated, which increases the oxidative stress and free

radical production, thereby causing DNA damage (Doull, Cattley et al. 1999). It is

also assumed that peroxisome proliferators increase cell proliferation and inhibit

apoptosis, which may also contribute to cancer development (Tickner, Schettler et al.

2001).

However, relevance of theses studies for humans is highly unlikely, because

humans express far less Ppar-α (1-10% of the level found in rodents (Palmer, Hsu et

al. 1998)). There are also genetic variations of human Ppar-α, which render it less

active compared to the rodent form (Palmer, Hsu et al. 1998; Woodyatt, Lambe et al.

1999). This is in line with the observation in large numbers of patients which are

treated with peroxisome proliferating drugs (e.g. hypolipidemic drugs), who show no

increased cancer incidence (Doull, Cattley et al. 1999). Additionally, studies with

Ppar-α (-/-) mice found no hepatic carcinomas after DEHP administration (Peters,

Cattley et al. 1997). However, these mice exhibited the remaining adverse effects

attributed to DEHP: testicular lesion, kidney effects and fetotoxicity (Peters,

Taubeneck et al. 1997; Ward, Peters et al. 1998).

2.2.3.4 Reproductive and Developmental Toxicity

There have been multitudes of studies analysing the developmental and

reproductive toxicity of DEHP and its metabolites. As an extensive discussion of all

studies would be beyond the scope of this thesis, only a relevant subset is discussed

below. The additional studies are compiled in the risk assessment report on DEHP by

the European Chemical Bureau (ECB) (Bureau 2003) and the risk assessments by

the US Agency for Toxic Substances and Disease Registry (ATSDR) and

Environmental Protection Agency (EPA) (Kavlock, Barr et al. 2006).

DEHP affects fertility and reproduction of both sexes; it also interferes with the

development of the offspring. While other phthalates also possess some estrogenic

activity, the reproductive toxicity of DEHP is the highest (Heindel, Gulati et al. 1989).

Repeated exposure to DEHP induced testicular toxicity in male rats and mice. The

effects included: reduced testis weight, reduced sperm production, reduced

testosterone production, seminiferus tubular atostrophy, vacuolisation of Sertoli cells

and undescended testis (Gray and Butterworth 1980; Shiota and Nishimura 1982;

Lamb, Chapin et al. 1987; Tyl, Price et al. 1988; Poon, Lecavalier et al. 1997; Gray,

Ostby et al. 2000; Moore, Rudy et al. 2001; Wolfe 2003; Andrade, Grande et al.


18

2006; Dalsenter, Santana et al. 2006). Non-human primates seem to be less

sensitive towards DEHP toxicity. Exposure of up to 2500 mg/kg bw per day did not

influence the testicular development in male marmosets (Tomonari, Kurata et al.

2006).

These toxic effects were especially severe when animals were exposed in utero

and/or before they were sexually mature. The lowest reported LOEL for

developmental toxicology for in utero exposure and during suckling are as low as

3.5 mg/kg bw per day (Arcadi, Costa et al. 1998). The current NOELs which are

selected for the human risk characterisation are the 4.8 mg/kg bw per day (Wolfe

2003) or 3.7 mg/kg bw per day (Poon, Lecavalier et al. 1997).

The main targets of DEHP - or rather its metabolite MEHP - in male animals are

the Leydig and Sertoli cells. Sertoli cells provide support for the germ cells and

respond to follicle stimulating hormone (FSH). FSH is necessary for the initiation and

maintenance of spermatogenesis. The main targets in female animals are the

granulosa cells (the equivalent of Sertoli cells in males). MEHP prevents the FSH

stimulation of granulosa cells in vitro. This leads to decreased estradiol production,

prevents ovulation and prolongs the estrous cycle (Reviewed in Lovekamp-Swan and

Davis 2003).

This hormone pathway is analogous in humans and rodents. It is therefore

reasonable to assume that DEHP does also affect humans. Limited studies on the

effect of DEHP on human populations suggest that occupational exposure to DEHP

via PVC increases the risk for testicular cancer (Hardell, Ohlson et al. 1997). Higher

levels of phthalates in human serum are also correlated with increased pregnancy

complications, decreased pregnancy rates, endometriosis (Cobellis, Latini et al.

2003), short angogenital distance in male offspring (Swan, Main et al. 2005) and

abnormal reproductive development (Colon, Caro et al. 2000).

2.2.4 Concerns

At the moment there is a controversial debate about the danger of phthalates as

endocrine disruptors in humans (Lottrup, Andersson et al. 2006; Marsee, Woodruff et

al. 2006; McEwen and Renner 2006; Queiroz and Waissmann 2006). In order to limit

the risks DEHP has been banned from use in children’s toys and personal care

products (e.g. cosmetics, lotions, perfumes) within the EU since 2005

A Introduction Uremic Toxins

19

(Umweltbundesamt 2006). The discussion weather DEHP should be or can be

replaced in medical devices is still ongoing.

3 Uremic Toxins

As renal failure progresses and renal clearance declines, compounds which are

normally excreted begin to accumulate in the blood. A number of these retention

solutes exhibit toxic properties. Additionally they can become substrates for further

biological reactions in the uremic milieu and can thereby contribute to the adverse

effect (Himmelfarb, Stenvinkel et al. 2002). These substances are called uremic

toxins.

A uremic toxin has to meet the following criteria:

• it is a chemical or biological agent capable of producing a response

• it interacts with the biological system and produces a biological response

• the response should be considered deleterious to the biological system

(Vanholder, Argiles et al. 2001)

At the moment more than 90 uremic toxins are known but the number is

increasing constantly (Tab. A-4) (Vanholder, De Smet et al. 2003). The uremic toxins

differ in their molecular weight and their binding capacity to proteins. There are free

water soluble low molecular-weight solutes like urea [MG < 500 Da]; protein bound

toxins like homocysteine (Hcy) and advanced glycation end-products (AGEs) [MW

mostly < 500 Da]; and middle molecules like tnf-α or leptin [MW 500 - 15,000 Da]

(Vanholder, De Smet et al. 2003).

Some of the uremic toxins which are suspected to possess genotoxic capacity

are discussed below.


20

Uremic toxins:

Free water-soluble low-MW solutes e.g.:

• Creatinine

• Cytidine

• Mannitol

• Methylguanidine

• Oxalate

• Urea.....

• …

Protein-bound solutes e.g.:

• Homocysteine

• Indoxyl sulfate

• Indole-3-acetic acid

• Leptin

• Methylglyoxal (AGE)

• …

Middle molecules e.g.:

• β2-microglobulin

• Interleukin-6

• Neuropeptide Y

• Tumor necrosis factor-α

• …

Tab. A-4 Examples of uremic toxins

(Vanholder, De Smet et al. 2003)

3.1 Homocysteine

3.1.1 Chemical Structure and Pathways

Homocysteine (Hcy; 2-amino-4-mercaptobutyric acid) is an analogue of the

essential, sulphur-containing amino acid cysteine (Fig. A-6).

CH

CH2

CH2

SH

COOH

NH2

Fig. A-6 The chemical structure of homocysteine

It can be metabolised by two pathways: the transsulfuration sequence or the re-

methylation cycle (Finkelstein 1990) (Fig. A-7). The re-methylation cycle outweighs


21

the transsulfuration sequence in most mammalian cells. In this pathway Hcy is re-

methylated to methionine (Met) by the vitamin-B12 dependent methylfolate-

homocysteine methyltrasferase, using 5-methyltetrahydrofolate as methyl donor. The

generated Met is converted to S-adenosylmethionine (SAM) – a methyl donor for

DNA and proteins. SAM takes part in numerous specific transmethylation reactions

which yield S-adenosylhomocysteine (SAH) as a product. Finally the adenosyl-

homocysteinase uses SAH to synthesize Hcy (Finkelstein and Martin 2000).

Apart from this basic re-methylation cycle, Hcy can also be methylated by a

second Hcy-methylase, which employs betaine as methyl donor. This Hcy-methylase

is primarily found in the liver of all mammalian species and in the primate kidney

(McKeever, Weir et al. 1991).

The transsulfuration pathway leads to the irreversible removal of Hcy from the

organism. At first, the cystationine-β-synthetase (vitamin B6-dependent) catalyses the

reaction of Hcy with serin. In this process cystathione is formed. Ammoniac and α-

ketobtyrate are cleaved from cystathione by the cystathionine-γ-lyase forming

cysteine (Lehninger, Nelson et al. 1994). Cysteine is an important precursor for the

synthesis of the cellular antioxidant glutathione. The transsulfuration pathway is

found primarily in the liver, kidney, pancreas and intestine (Finkelstein 1990). Apart

from participating in these two pathways, Hcy can also form the reactive compound

homocysteine-thiolactone (Hcy-T) (s. 3.1.6, page 27).


22

5,10 Methylene THF

MET

Hcy

SAM

Methylation of e.g. proteins, DNA, RNA

THF

5-Methyl THF

Folate

Dihydrofolate

SAH

Cystathionine

Cysteine

Glutathione

Hcy-T

Dietary protein

DMG

Betaine

R

R-CH3

Vit B6

Vit B12

MSBHMT

Vit B6

Serine

Ketobutyrate + NH4+

MTHFR

MAT

AH

CBS

5,10 Methylene THF

MET

Hcy

SAM

Methylation of e.g. proteins, DNA, RNA

THF

5-Methyl THF

Folate

Dihydrofolate

SAH

Cystathionine

Cysteine

Glutathione

Hcy-T

Dietary protein

DMG

Betaine

R

R-CH3

Vit B6

Vit B12

MSMSBHMTBHMT

Vit B6

Serine

Ketobutyrate + NH4+

MTHFR

MATMAT

AH

CBS

Fig. A-7 Hcy metabolism and the enzymes and vitamins involved

Hcy is an intermediate in the sulfur amino acid metabolism. It is linked with the

methionine cycle (left) and the folate cycle (right). It can finally be removed from

these cyles via the transsulfuration pathway.

Abbreviations: AH, adenasyl-homocysteinase; BHMT, betaine-homocysteine-S-

methyltransferase; CBS, cystathione-β-synthase; Hcy, homocysteine; Hcy-T,

homocysteine-thiolactone; MAT, methionine-adenosyltransferase; MET,

methionine; MS, methionine synthase; MTHFR, methylenetetrahydrofolate

reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF,

tetrahydrofolate

Intracellularly, Hcy is present in its free and reduced forms but the concentration

remains within strict limits. The main regulatory mechanism for maintaining the Hcy

equilibrium in the cell is via export into the plasma (Reviewed in Blom and De Vriese

2002).

After entering the bloodstream Hcy is rapidly oxidized. Seventy percent are

bound to proteins mainly serum albumin the rest occurs as homocystin - the disulfide

of homocysteine - and homocysteine-cysteine mixed disulfides, while only 1 - 2%

exist as free, reduced thiol (Finkelstein and Martin 2000).


23

3.1.2 Homocysteine Levels

The normal plasma concentration of total homocysteine is between 8 – 10 µM in

females and 10 - 12 µM in males (Perna, Ingrosso et al. 2003). Mild (12 - 15 µM) to

moderate hyperhomocysteinemia (16 - 30 µM) is found in 5 - 10% of the population

(Stanger, Herrmann et al. 2003). Values between 31 – 100 µM are regarded as

intermediate hyperhomocysteinemia and values > 100 µM as severe

hyperhomocysteinemia (Perna, Ingrosso et al. 2004). The plasma levels of Hcy

increase with age (Hernanz, Fernandez-Vivancos et al. 2000) and are generally

higher in men than in women (Lussier-Cacan, Xhignesse et al. 1996). This is

probably an estrogenic effect as the gender difference disappears after menopause.

The intra-individual variability is very low and no seasonal effect could be determined.

One group of persons in which hyperhomocysteinemia is very common are

patients suffering from ESRD (Suliman, Qureshi et al. 2000; Mallamaci, Zoccali et al.

2002; Kalantar-Zadeh, Block et al. 2004; Wrone, Hornberger et al. 2004; Nair,

Nemirovsky et al. 2005; Perna, Satta et al. 2006). Roughly 90% of these patients

suffer from hyperhomocysteinemia, mostly moderate to intermediate

hyperhomocysteinemia, although severe cases are not uncommon.

The plasma Hcy level strongly correlates with the glomerula filtration rate, but the

precise mechanism is not definitely established (van Guldener 2006). Unlike in many

healthy, but folate deficient persons, the administration of folic or folinic acid does not

normalize the Hcy levels in HD patients (Armada, Perez et al. 2003). However, it still

reduces the Hcy level.

3.1.3 Reasons for Elevated Homocysteine Levels

The reasons for hyperhomocystemia in ESRD patients are complex and still not

completely understood. Impaired renal excretion of Hcy was thought to be the

underlying reason, but as the amount of Hcy in the urine is only about 6 µmol/l this

seems unlikely (Refsum, Helland et al. 1985). Another hypothesis is that uremic

toxins impair some of the enzymes, relevant to the Hcy metabolism (van Guldener

and Stehouwer 2003; Perna, Ingrosso et al. 2004). This is in line with the observation

of a decreased remethylation and transmethylation flux in ESRD patients, while the

transsulfuration rate of those patients is similar to control subjects (van Guldener,

Kulik et al. 1999; van Guldener 2006). Furthermore, uremic patients, with their poor

appetites and recommended low protein diets, are under constant duress to produce


24

enough methyl groups to sustain the normal transmethylation rate they need to

prevent the accumulation of Hcy. The same holds true for the vitamin supply of cells,

especially as the transmembrane transport of folate is impaired in ESRD patients

(Jennette and Goldman 1975).

Apart from ESRD, gene mutations, reduced vitamin intake or intestinal absorption

as well as the intake of certain drugs can also lead to increased Hcy levels (Tab.

A-5).


25

Causes/determinants

Genetic factors:

• Homocystinuria

• Heterozygosity for CBS defects

• Down syndrome

• MTHFR 677C→T (homozygosity)

• Other polymorphisms

Physiologic determinants:

• Increasing age

• Male sex

• Pregnancy

• Postmenopausal state

• Renal function, reduced GFR

• Increasing muscle mass

Lifestyle determinants:

• Vitamin intake (folate, B12, B6, B2)

• Smoking

• Coffee

• Ethanol

• Exercise

Clinical condition:

• Folate deficiency

• Cobalamin deficiency

• Vitamin B6 deficiency

• Renal failure

• Hyperproliferative disorders

• Hypothyroidism

• Hyperthyroidism

• Diabetes

Drugs:

• Lipid lowering (cholestyramine, fibric acid derivates, nicotinic acid)

• Anticonvulsants (phenytoin, carbamazepine)

• Sex hormones (androgens)

• Anti-rheumatic drugs (methotrexate)

• Other (cyclosporin, diuretics, levodopana)

Tab. A-5 Determinants of plasma total Hcy

(Hankey, Eikelboom et al. 2004)

3.1.4 Clinical Implications of Elevated Homocysteine Levels

The first researcher to suggest adverse effects of Hcy was McCully in 1969. He

proposed that elevated levels of Hcy cause arteriosclerosis (McCully 1969). Since

then, elevated levels of total Hcy have also been related to birth defects and


26

pregnancy complications (Miller and Kelly 1996; Vollset, Refsum et al. 2000) as well

as psychiatric disorders (Nilsson, Gustafson et al. 1996) and cognitive impairment in

the elderly (Dimopoulos, Piperi et al. 2006; McMahon, Green et al. 2006).

Most importantly, increased plasma concentrations of total Hcy have been

regarded as strong and independent risk factors for cardiovascular disease and

stroke (Nygard, Nordrehaug et al. 1997; Vollset, Refsum et al. 2001; Collaboration

2002; Wald, Law et al. 2002). However, within the last few years three large and well-

conducted prospective studies have set off a controversy whether total Hcy is a risk

factor or merely an innocent bystander of the disease (Bonaa, Tverdal et al. 2006;

Craen, Stott et al. 2006; Khare, Lopez et al. 2006; Lonn 2006; Quinlivan and Gregory

2006; Refsum and Smith 2006): the Heart Outcome Prevention Evaluation 2 (HOPE-

2) (Lonn, Yusuf et al. 2006), the Vitamin Intervention for Stroke Prevention trial

(VISP) (Toole, Malinow et al. 2004) and the Norwegian Vitamin trials (NORVIT)

(Bonaa, Njolstad et al. 2006). All studies included several thousand participants

(HOPE-2: 5522, VISP: 3680 and NORVIT: 3749) and showed that lowering of tHcy

by administration of B vitamins and/or folate did not reduce the risk of cardiovascular

events or stroke compared to a placebo group. However, these studies covered only

a two to five year period, therefore one cannot rule out that a positive effect might be

observed at a later date.

Conflicting results have been reported for total Hcy and mortality in ESRD

patients. While some studies report a strong association between

hyperhomocysteinemia and cardiovascular mortality (Bostom, Shemin et al. 1997;

Moustapha, Naso et al. 1998; Mallamaci, Zoccali et al. 2002), others could not

confirm this association (Menon, Sarnak et al. 2006) or even reported an inverse

relationship (Suliman, Qureshi et al. 2000; Kalantar-Zadeh, Block et al. 2004; Wrone,

Hornberger et al. 2004; Nair, Nemirovsky et al. 2005; Ducloux, Klein et al. 2006).

Additionally, high total Hcy levels have even been discussed as new tumour

marker (Wu and Wu 2002)

3.1.5 Hyperhomocysteinemia and Cancer

Several studies have found a significant positive correlation between elevated

levels of total Hcy and increased MN frequency in healthy men between 50 and 70

(Fenech, Dreosti et al. 1997; Fenech 1999). This observation was also true for young

Australian males [18 - 32 years], but not females (Fenech, Aitken et al. 1998). These


27

correlations are supported by preliminary in vitro studies on human lymphocytes.

There was a moderate increase of MN in cells treated with 50 – 400 µM Hcy (Crott

and Fenech 2001).

Further evidence for the involvement of Hcy in cancer development is its

influence on DNA cytosine-methylation; however the results of these studies are

conflicting. One study on patients with chronic alcoholism correlated Hcy levels with

DNA hypermethylation in peripheral blood cells of patients (Bonsch, Lenz et al.

2004), while a small study on ESRD patients found that plasma total Hcy

concentration correlated significantly with DNA hypomethylation (Ingrosso, Cimmino

et al. 2003).

Oxidative stress may also lead to DNA damage. Although all dialysis patients

suffer from increased oxidative stress, the discussed whether Hcy contributes is still

ongoing (Bayes, Pastor et al. 2001; Bayes, Pastor et al. 2003).

The same holds true for in vitro tests; while Hcy elicited oxidative stress in some

test systems (Austin, Sood et al. 1998; Au-Yeung, Woo et al. 2004; Perez-de-Arce,

Foncea et al. 2005), it did not in others (Outinen, Sood et al. 1998; Lynch, Campione

et al. 2000; Zappacosta, Mordente et al. 2001) or had antioxidant as well as pro-

oxidant effects (Lynch and Frei 1997).

3.1.6 Homocysteine-Thiolactone

Homocysteine-thiolactone (Hcy-T) is formed in human cells by the enzymatic

conversion of Hcy to its corresponding thioester. This conversion occurs when Hcy

falsely enters the protein biosynthetic apparatus because of its similarity to

methionine, isoleucin and leucin. However, Hcy cannot complete the biosynthetic

pathway (Jakubowski 2004).

After misactivation by methionyl-tRNA to Hcy-AMP (thereby using ATP), Hcy-

AMP is subsequently destroyed by the enzyme forming a cyclic thioester - Hcy-T

(Fig. A-8). This reaction is universal to all cells and prevents the misincorporation of

Hcy into proteins (Jakubowski 1997). Hcy-T can be reconverted to Hcy by enzymes

like the intracellular bleomycin hydrolase or the extracellular HDL-associated - Hcy-

thiolactonase (Jakubowski 2006).


28

AMP

THcyPPAMPHcyAARATPHcyAARS i

−

↓

−⇒+•⇔++ ~

Fig. A-8 Equation for the formation of Hcy-T (Jakubowski 1999)

AARS: aminoacyl-tRNA; Hcy: homocysteine; ATP: adenosine triphosphate;

AMP: adenosine monophosphate; ppi: pyrophosphate; Hcy-T: homocysteine-

thiolactone

The energy of the anhydride bond Hcy∼AMP is conserved in an intra-molecular

thioester bond of Hcy-T. Consequently, Hcy-T is chemically reactive and acetylates

free aminogroups easily (Jakubowski 1999).

Under physiological conditions Hcy-T is neutral in charge and freely diffuses

through cell membranes (Jakubowski and Goldman 1993; Jakubowski 1997) (Fig.

A-9). Therefore it is assumed that increasing levels of Hcy lead to increasing

misactivation, finally leading to the formation of Hcy-T, which then leaves the cell.

Fig. A-9 Hcy/Hcy-T metabolism (adapted after Jakubowski 2006)

MetRS, methionyl-tRNA synthetase; BLH, bleomycin hydrolase; HDL, HDL-

associated -Hcy-thiolactonase


29

In plasma Hcy-T undergoes two major reactions: (1.) acylation of aminogroups in

proteins (primarily ε-amino groups of lysine residues) and (2) enzymatic hydrolysis to

give Hcy, which then attaches to proteins by forming a protein-S-S-Hcy disulfide

(Jakubowski 1999). This may lead to protein damage.

The chemical reactivity of Hcy-T leads to a half-life of approximately 1 h in blood

and plasma (Jakubowski 1999). In healthy subjects the Hcy-T levels vary between 0

– 34.8 nM (Daneshvar, Yazdanpanah et al. 2003; Chwatko and Jakubowski 2005);

representing about 0 – 0.28% of tHcy. Surprisingly, there is no correlation between

total Hcy levels and Hcy-T concentration in plasma (Jakubowski 2006). This suggests

that it is not Hcy which is the major determinant of Hcy-T but HDL-associated-Hcy-

thiolactonase, methionyl-tRNA synthetase or renal excretion. In fact, Hcy-T is

efficiently eliminated by urinary excretion, with a Hcy-T concentration up to 100-fold

higher than in plasma (Chwatko and Jakubowski 2005).

3.2 Advanced Glycation End-Products (AGEs)

3.2.1 Formation of Advanced Glycation End-Products

AGEs are a heterogeneous group of molecules. Six AGEs (fructoselysine,

carboxymethyllysine, pyrraline, pentosidine, glyoxal-lysine dimer, methylglyoxxal-

lysine-dimer) are classified uremic toxins (Vanholder, Argiles et al. 2001; Vanholder,

De Smet et al. 2003). They are generated by the non-enzymatic reaction of reducing

sugars and the free amino groups of individual amino acids, peptides or proteins. The

unstable Schiff’s base which is formed in this process can isomerise and form the

Amadori product. A subsequent series of complex biochemical reactions like

dehydration, condensation, fragmentation and oxidation slowly leads from Amadori

product to final AGEs (Fig. A-10), (Reviewed in Bohlender, Franke et al. 2005;

Sebekova, Wagner et al. 2007).


30

Fig. A-10 Formation of AGEs (adapted after Raj, Choudhury et al. 2000)

This process occurs endogenously or during food processing (e.g. heating).

Actually, diet-derived and orally absorbed AGEs are suspected to contribute

significantly to the overall AGE load in renal failure patients (Koschinsky, He et al.

1997; Uribarri, Peppa et al. 2003; Goldberg, Cai et al. 2004). Independent of their

origin, AGEs are cleared by the kidney (Gugliucci and Bendayan 1996). Miyata et al.

showed the fate of an exemplary AGE – pentosidine: it is filtered by the glomeruli and

reabsorbed by the proximal tubule cells. There it is modified, degraded and

eventually excreted in the urine (Miyata, Ueda et al. 1998). Therefore AGE levels

correlate inversely with creatinine clearance, which leads to up to 6-fold elevated

AGE levels in ESRD patients (Makita, Radoff et al. 1991; Makita, Bucala et al. 1994).

Apart from reduced renal function, elevated plasma glucose may contribute to

elevated AGE levels (Makita, Radoff et al. 1991; Brownlee 1995).

3.2.2 Biological Effects of AGEs

Elevated levels of AGEs have been correlated to several diseases, e.g.

Alzheimer’s disease (Gasic-Milenkovic, Loske et al. 2003; Lue, Yan et al. 2005; de

Arriba, Stuchbury et al. 2007), arteriosclerosis (Turk, Sesto et al. 2003), and have

been related to the aging process (Brownlee 1995) and nephropathy.

The mechanisms by which AGEs induce pathological changes are still a focus of

ongoing research. One pathway is by interaction with the receptor for advanced

glycation end products (RAGE). RAGE is a cell surface receptor which belongs to the

immunoglobulin superfamily. Binding to this receptor leads to initiation of intracellular

hours days weeks

Glucose + R-NH2 Schiff

base Amadori

Product

Intermediate

glycosilation

products

AGEs

H O

\ //

C

|

(CHOH)4+NH2-R

|

CH2OH

H NH-R

\ //

C

|

(CHOH)4

|

CH2OH

CH2NH-R

|

C = O

|

(CHOH)3

|

CH2OH

HO

HO O

N

HO HO

OH

CHO

O

O

O

N

N

+


31

signalling cascades, including NF-κB activation resulting in inflammation and immune

response. This includes macrophage activation, increased cytokines, chemokines,

growth factors and ROS expression (Yan, Schmidt et al. 1994; Wendt, Tanji et al.

2003 reviewed in Schmidt, Yan et al. 2001; Lin 2006). As the RAGE expression is

upregulated under high AGE conditions these reactions enhances themselves (Hou,

Ren et al. 2004).

Additionally AGEs have been shown to exert genotoxic effects in vitro (Bucala,

Model et al. 1985; Mullokandov, Franklin et al. 1994; Murata, Mizutani et al. 2003;

Roberts, Wondrak et al. 2003; Schupp, Schinzel et al. 2005) and may therefore be

involved in the cancer development of ESRD patients.

3.3 Leptin

Leptin, the16 kDa product of the ob gene (obese gene), was discovered in 1994

by Zhang et al. (Zhang, Proenca et al. 1994). The 167 amino acid hormone is mainly

produced by adipocytes and has gained interest because of its involvement in the

regulation of food intake and energy expenditure. Today it is known that leptin acts

as a multifunctional hormone (Fruhbeck 2006) and influences the immune system

(Lam and Lu 2007), female and male reproduction, the mammary glands, the gut, the

kidney and the lungs (Baratta 2002) as well as blood pressure (Fruhbeck 1999),

bone formation and angiogenesis.

Leptin exerts its physiological function through the binding to Ob-receptors, which

belong to the cytokine class 1. To date, six isoformes of the leptin receptor are

known, which are generated by alternative splicing of the db gene (Harvey 2007).

Those receptors have identical extracellular and transmembrane domains but differ in

their intracellular domain. The Ob-Rb is the most important receptor, because it is the

only one which is fully functional and can submit a signal via the JAK/STAT (Janus

kinases/signal transducers and activators of transcription) or MAPK (mitogen-

activated protein kinase) pathways. The main function of the other receptors is

probably transport of leptin through the brain barrier and uptake of leptin for

degradation (Huang and Li 2000; Chelikani, Glimm et al. 2003; Hegyi, Fulop et al.

2004).

Plasma levels of leptin vary between 11.9 ± 3.1 µg/l (males) and 21.2 ± 3 µg/l

(females), being higher in females than in males, and are closely related to the

amount of body fat and BMI (Horn, Geldszus et al. 1996).

A Introduction Cancer

32

3.3.1 Leptin – an Uremic Toxin?

In many (Merabet, Dagogo-Jack et al. 1997; Sharma, Considine et al. 1997) but

not all (Stenvinkel, Heimburger et al. 1997) ESRD patients, leptin levels are

enhanced by the 2 - 4 fold. However, only the level of free leptin in the plasma is

increased, while the protein bound fraction remains stable (Widjaja, Kielstein et al.

2000). In spite of the elevated leptin levels, ESRD patients have significantly lower

leptin mRNA expression compared to BMI matched controls. This is possibly due to a

negative feedback regulation by decreased renal clearance (Nordfors, Lonnqvist et

al. 1998).

Due to the multifunctional nature of leptin, it is easy to imagine that elevated

levels may have a negative impact on the patient. Still, leptin is only classified as

suspected uremic toxin (Vanholder, De Smet et al. 2003). However, the evidence for

negative impact of leptin in dialysis patients is increasing. Because leptin reduces

appetite and increases the metabolic rate, it has been speculated whether leptin

might be one of the factors that mediates anorexia and wasting syndrome in ESRD

patients. So far the study results are controversial: some fail to find a correlation

between nutritional markers and hyperleptinemia (Koo, Pak et al. 1999; Rodriguez-

Carmona, Perez Fontan et al. 2000), while others find an inverse correlation (Young,

Woodrow et al. 1997; Johansen, Mulligan et al. 1998).

It has been shown that leptin stimulates proliferation and differentiation of

hemopoietic cells (Gainsford, Willson et al. 1996), and acts as an act anti-apoptotic

(Konopleva, Mikhail et al. 1999, Artwohl, 2002) thereby promoting the proliferation of

colorectal cancer cells(Rouet-Benzineb, Aparicio et al. 2004; Ogunwobi and Beales

2007). It also promoted the proliferation of prostate cells (Deo, Rao et al. 2008). In

support of these in vitro findings, leptin levels of HD patients were correlated with

peripheral DNA damage detectable by comet assay (Horoz, Bolukbas et al. 2006).

4 Cancer

Cancer is a complex disease in which altered gene expression leads to abnormal

cell proliferation and invasion of other tissues, thereby disrupting their normal

function. There are several theories about the cause of cancer but all of them

assume that several critical mutations have to take place until a cancer phenotypic

cell is formed (Fenech 2002). Critical mutations are located e.g. within apoptosis


33

genes, oncogenes, tumorsuppressor genes, mismatch repair genes or cell cycle

control genes.

Given that an accumulation of mutations cannot be explained by single point

mutations, it has been suggested that mutations lead to hypermutatbility, which later

initiates cancer (Cahill, Kinzler et al. 1999; Tomlinson 2001; Coleman and Tsongalis

2006).

One possible mechanism which converts a cell that way is by generation of

aneuploidy (abnormal number of chromosomes due to loss or gain of one or more

chromosome) (Li, Sonik et al. 2000; Fenech 2002). According to this hypothesis the

generation of cancer cells follows two steps: (1) a carcinogen leads to aneuploidy; (2)

aneuploidy destabilizes the karyotype finally leading to neoplastic karyotypes (Li,

Sonik et al. 2000).

Another factor which is very likely to increase the mutation frequency is direct

DNA damage. Indeed, several well-documented studies correlate DNA damage and

cancer in laboratory animals as well as in humans (Hagmar, Bonassi et al. 1998).

4.1 Types of DNA Damage

There are several types of DNA damage: single strand breaks, mismatch of

bases, hydrolysis of bases, alkylation of DNA, DNA cross-linking, DNA oxidation and

changes in DNA methylation patterns. The types relevant for the present thesis are

discussed below.

4.1.1 DNA Oxidation

DNA oxidation occurs when the DNA is attacked by reactive oxygen species

(ROS). ROS are produced e.g. during cellular respiration, which involves the

reduction of O2 to H2O in the electron transport chain. Intermediates of this process

are ROS like O2-, H2O2 and (HO⋅). If a substance interferes with the electron

transport chain or disrupts the mitochondrial membrane, some of these ROS can

escape the electron transport chain. Additionally, several oxidizing enzymes like e.g.

NADPH-oxidase, as well as toxins or radiation can produce ROS.

Under normal circumstances, ROS are converted to water and molecular oxygen

by the enzymatic antioxidant defence system of the cell. However, if there is an

imbalance between antioxidants and ROS (oxidative stress), ROS - especially

hydroxylradicals - can attack the nitrogenous bases or the sugar-phosphate-


34

backbone of the DNA. This leads to hydroxylation, ring opening and fragmentation

(Sies 1997; Kelly, Havrilla et al. 1998; Griendling, Sorescu et al. 2000; Vaziri 2004).

Normally this is repaired but if the damage is not recognised or severe this can lead

to mutations.

Therefore it is not surprising that numerous studies link oxidative stress to cancer

(Bendesky, Michel et al. 2006; Beevi, Rasheed et al. 2007; Hori, Oda et al. 2007;

Nayak and Pinto 2007). Additionally, ROS can also react with proteins, lipids and

carbonhydrates, thereby causing inflammation, apoptosis, fibrosis and cell

proliferation.

4.1.2 Changes in DNA Cytosine-Methylation

DNA methylation is the epigentic modification of DNA by the covalent addition of

a methyl group on the 5’ carbon of cytosine within the context of the CpG nucleotide.

DNA methylation patterns are established by at least three independent

methyltransferases. Approximately 70% of the DNA is methylated (Robertson and

Jones 2000). However, methylation patterns are not random but heritable, tissue-

and species specific (Kim 2004). DNA methylation is an important factor for gene

expression, DNA conformation and stability, binding of transcription factors, x-

chromosome inactivation and, imprinting and suppression of parasitic DNA

sequences (Kim 1999; Robertson and Jones 2000). In this process methylated DNA

sequences become transcriptionally silenced (Robertson and Jones 2000).

Due to their big impact on DNA it is not remarkable that changes in DNA

methylation patterns have been associated with several cancers, e.g. breast cancer

(Widschwendter and Jones 2002), lung cancer (Piyathilake, Frost et al. 2001),

colorectal cancer (Kim 2004) and kidney cancer (Cairns 2004). Generally, the cancer

genome can be characterized by hypermethylation of specific genes and a

simultaneous overall decrease of 5-methyl-cytosine (Zhu and Yao 2007). The degree

of overall DNA methylation decreases progressively during the stages of neoplasia

from benign proliferation to invasive cancer (Fraga, Herranz et al. 2004).

B Objectives

35

B Objectives

The objective was to examine the role of toxins in hemodialysis therapy and their

possible contribution to the increased genomic damage of HD patients. Two possible

sources for those toxins have been considered: (1) substances leaching from the

extracorporal blood circuits, and (2) uremic toxins accumulating in the blood of HD

patients.

The first part of this work focused on substances leaching from dialysers and

tubing. Therefore eluates of different dialysers under conditions similar to the ones

during dialysis had to be produced. Next, the substances leaching from dialyser into

the eluates had to be analysed by LC-MS/MS. In order to examine the contribution of

those substances to the genomic damage of HD patients, cytotoxicity and

genotoxicity of those eluates had to be by analysed by in vitro studies. As some

cancers are responsive to xenoestrogens, the estrogenic activity of eluates had to be

examined by E-Screen.

The second part of the work focused on the toxicity of accumulated uremic

toxins. As more than 90 uremic toxins are known, a sensible selection of the uremic

toxins tested had to be made. We chose Hcy because elevated Hcy levels have been

correlated to increased MN frequency and because it induced MN formation in vitro.

In order to estimate the relevance of this genomic damage to the dialysis patients,

possible mechanisms for MN induction had to be analysed. Frequent reasons for

DNA damage are oxidative stress, changes in DNA methylation or disturbances

during mitosis. Therefore those possibilities had to be analysed. Additionally the

genotoxicity of Hcy-T, leptin and AGEs was analysed.

C Materials & Methods General Materials

36

C Materials & Methods

1 General Materials

1.1 General Technical Equipment:

The following equipment was used for the experiments (Tab. C-1).

Product Manufacturer

Autoclave Melag Autoklav 23, Berlin, Germany

Camera Cohn-High Performance, CDD Camera, INTAS Science Imaging Instruments, Göttingen, Germany

Centrifuges

Universal / K2S, Hettich, Tuttlingen, Germany

Laborfuge 400e, Heraeus, Hanau, Germany

Spectrafuge 24D, Abimed, Langenfeld, Germany

Universal 16R, Hettich, Tuttlingen, Germany

Coulter Counter Coulter Z1, Coulter Electronics, GB

Cytocentrifuge Cytospin 3, Shandon, GB

Flow cytometer BD FACS LSR, 2 Laser, H0108, Becton Dickinson, Heidelberg, Germany

Freezing Container NALGENETM

Cryo 1°C, Nalgene, USA, Cat. No: 5100-0001

Heating plate Gerhardt H22 electronic, Bonn, Germany

HPLC Agilent 1100, Agilent Technologies, Santa Clara, USA

Incubator Type B, 5060 EK-CO2, Heraeus, Germany

Laminar Flow Gruppo Flow, Gelaire BH26, Flow Laboratories, Germany

AntairBSK, BSK4, Antair

Linear ion trap mass spectrometer

QTrap, Applied Biosystems, Darmstadt, Germany

Microscopes

Fluorescence Microscope (comet assay): Nikon, Labphot 2 A/L, Nikon, Germany

Fluorescence Microscope (micronuclei): Zeiss, Jena, Germany

Light microscope (cell culture): Labovert, Leitz, Wetzlar, Germany

Microwave Oven M630, Phillips, Hamburg, Germany

Peristaltic pump Typ: BV-GE, ISMATEC Laboratoriumstechnik GmbH, Wertheim-Mondfeld, Germany

pH meter pH 526, Multical WTW, Weilheim, Germany

Photometer Hitachi, U-2000, Tokyo, Japan

Pipettes Gilson, Middleton, USA

Eppendorf, Hamburg, Germany

Shaker KL2, Edmund Büchler, Germany

Sterilizer T 6120, Heraeus, Hanau, Germany

Triple quadrupole instrument

API 3000, Applied Biosystems, Darmstadt, Germany

Vortex Vortex Genie 2, Bender & Hobein, Zürich, Switzerland

Water Bath GFL, Type 1012, Gesellschaft für Labortechnik GmbH, Burgwedel, Germany

GFL, Type 1083, Gesellschaft für Labortechnik GmbH, Burgwedel, Germany

Tab. C-1 General technical equipment

C Materials & Methods General Materials

37

1.2 General Materials and Chemicals

Unless otherwise stated chemicals not listed in Tab. C-2 were purchased from

Sigma-Aldrich, Taufkirchen, Germany.

Product Manufacturer/Supplier

5 ml Multitips Multitips, sterile, Laborbedarf Hartenstein, Würzburg, Germany, Cat. No: 4003-0013

Acridine orange Serva, Heidelberg, Germany, Cat. No: 21572

Bio-Rad Protein Assay, Dye Reagent Concentrate

Bio-Rad, Munich, Germany, Cat. No: 500-006

Bovine Serum Albumine Sigma-Aldrich, Taufkirchen, Germany, Cat. No: A7906

Cell culture flasks Sarstedt, Nürnbrecht, Germany

Cryo-vials Greiner, Solingen-Wald, Germany, Cat. No: 121263

Cuvettes (10×4×45 mm) Sarstedt, Nürnbrecht, Germany, Cat. No: 67.704

Dialysis tubing membranes Sigma-Aldrich, Taufkirchen, Germany

DMSO (Dimethyl sulfoxide) Sigma-Aldrich, Taufkirchen, Germany, Cat. No: D4540

EDTA (Ethylendiamine-tetra aceticacid)

Roth, Karlsruhe, Germany, Cat. No: 8040.1

Ethanol Roth, Karlsruhe, Germany, Cat. No: P006.1

FACS Clean BD Biosciences, Heidelberg, Germany, Cat. No: 340345

FACS Rinse BD Biosciences, Heidelberg, Germany, Cat. No: 340346

FACS tubes BD Biosciences, Heidelberg, Germany, Cat. No: 343675

Falcon tubes Sarstedt, Nürnbrecht, Germany

Methanol AppliChem, Darmstadt, Germany, Cat. No: A0688

Methyl-Methane-sulfonate Sigma-Aldrich, Taufkirchen, Germany, Cat. No: M4016

NaAsO2 Sigma-Aldrich, Taufkirchen, Germany, Cat. No: S7400

NaCl AppliChem, Darmstadt, Germany, Cat. No: A1149

NaOH AppliChem, Darmstadt, Germany, Cat. No: A1551

Propidium iodide (PI) Sigma-Aldrich, Taufenkirchen, Germany, Cat. No: P4864

Slides Assistent-Objektträger "ELKA", Glasfabrik Karl Hecht KG, Sondheim, Germany, Cat. No: 2406

Sodium acetate Sigma-Aldrich, Taufkirchen, Germany, Cat. No: S5636

Sterile filter Whatman® Sigma-Aldrich, Taufkirchen, Germany, Cat. No: F8552

Super frost slides AMNZ SuperFrost®Plus, Menzl-Gläser, Braunschweig, Germany, Cat. No: J1800

TRIS (Tris-hydroxymethyl-aminomethan)

Roth, Karlsruhe, Germany, Cat. No: A411.1

Triton X-100 Sigma-Aldrich, Taufkirchen, Germany, Cat. No: 93443

Tween 20 Sigma-Aldrich, Taufkirchen, Germany, Cat. No: P2287

Tab. C-2 General materials and chemicals

C Materials & Methods Cell Culture

38

2 Cell Culture

2.1 Media, Supplements and General Buffer

Media Manufacturer/Supplier

Dulbecco´s modified Eagle´s medium (DMEM) Sigma-Aldrich, Taufkirchen, Germany,

Cat. No: D5546

RPMI 1640

(With NaHCO3, without phenol red)

Sigma-Aldrich, Taufkirchen, Germany,

Cat. No: R7509

RPMI 1640

(With NaHCO3 and phenol red)


Cat. No: R0883

Horse serum Biochrom AG, Berlin, Germany, Cat. No: 59135

L-glutamine (stock solution 200 mM) Sigma-Aldrich, Taufkirchen, Germany,

Cat. No: G7513

Penicillin-Streptomycine (10000 U/ml penicillin, 10mg/ml streptomycine)


Cat. No: P0781

Sodium pyruvate (stock solution 100 mM) Sigma-Aldrich, Taufkirchen, Germany,

Cat. No: S8636

Accutase Sigma-Aldrich, Taufkirchen, Germany,

Cat. No: A6964

Trypsin-EDTA (stock solution: 10 x; EDTA: 2 g/l Trypsin: 5 g/l),


Cat. No: T4174

Fetal Bovine Serum (FBS) Biochrom, Berlin, Germany, Cat. No: S0115

Tab. C-3 Media and supplements

Name Ingredients

10 × PBS (Phosphate buffered saline)

1.4 M NaCl

27 mM KCl

100 mM KH2PO4/K2HPO4

pH 7.5

diluted to 1 × PBS with ddH2O prior to use

Tab. C-4 General buffer


39

2.2 Cell Lines, Media and Growth Conditions

Cell line Cell type Origin Medium

HL-60 Human acute myeloid leukemia

Established from the blood of a 35-year-old female with acute myeloid leukaemia in 1976

RPMI 1640 (R0883)

10% (v/v) FBS

1% (v/v) L-glutamine

0.4% (v/v) Penicillin-Streptomycine

MCF-7 Human breast adenocarcinoma

Established from the pleural effusion of a 69-year-old Caucasian female with metastatic mammary carcinoma (after radio- and hormone therapy) in 1970; cells were described of being positive for cytoplasmic estrogen receptors and having the capability to form domes (DSMZ 2004)

RPMI 1640 without phenolred (R7509)

10% (v/v) FBS


1 mM Sodium-pyruvate


L5178Y Mouse T cell lymphoma

Established from an 8-month-old female DBA/2 mouse with T cell lymphoma in 1985 (DSMZ 2004)

RPMI 1640 (R0883)

10% (v/v) PS


1% (v/v) Sodium-pyruvate


LLC-PK1 Porcine proximal tubule kidney cells

Established from the kidney of a male 17lb-Hamshire-pig (Hull, Cherry et al. 1976; Perantoni and Berman 1979)

DMEM

10% (v/v) FBS


2.5% (v/v) Hepes


CaCo-2 Human colonic carcinoma cell line

Established from the primary colon tumour (adenocarcinoma) of a 72-year-old Caucasian male in 1974 (Rousset 1986)

DMEM

10% (v/v) FBS



Tab. C-5 Cell lines, media and growth conditions

2.2.1 Maintenance of Cell Culture

Cells were grown in 75 cm2 flasks (20 ml medium) or in 25 cm2 flasks (5 ml

medium). The flasks were kept in an incubator with a moistened, 5% CO2

atmosphere at 37° C.

The medium was changed 3 times a week, generally on Monday, Wednesday

and Friday. Unless morphological or proliferation changes could be observed, cells

were used for experiments until passage 20.


40

2.2.2 Passaging of Cells

1. Adherent Cell Lines

Adherent cell lines like MCF-7, LLC-PK1 and CaCo-2 (see table C-5) were grown

until 80% confluency was reached. Then, cells were washed twice with 10 ml pre–

warmed PBS. PBS was removed and 2 ml of pre-warmed 1×Trypsin-EDTA were

added. After 2-10 minutes in the incubator (5% CO2; 37°C) cells started to detach.

The trypsin digestion was stopped by adding of 8 ml medium. Subsequently cells

were resuspended several times.

To obtain a new sub-culture, 2 ml of the cell suspension were transferred to a

new flask with 18 ml medium.

2. Non-Adherent Cells

In non-adherent cell lines like L5178Y or HL60 cells (see table C-5) the

concentration of cells was determined by coulter counter. For this purpose 200 µl of

the well mixed cell suspension were added to 9.8 ml isotone buffer. The number of

particles between 7.5 µm and 30 µM in a volume of 0.5 ml was determined by

counter.

The actual cell concentration was calculated as follows:

zC VDVnml

Cells⋅⋅⋅=

n = number of cells as determined by coulter counter

Vc= Sample volume in coulter counter

D = Dilution factor

Vz= Sample volume of the cell suspension

The volume containing the desired amount of cells (e.g. 1 × 106) was transferred

to a new flask.

2.2.3 Thawing of Cells

The cryo-vial containing frozen cells was taken out of the liquid nitrogen and

thawed within 2-4 minutes. The thawed cells were transferred to a 75 cm2 cell culture

flask filled with 20 ml cold medium. Cells were kept in an incubator (37°C; 5% CO2)

overnight. The next morning, cells were supplied with fresh medium.


41

2.2.4 Freezing of Cells

The freezing procedure was identical for all cell lines used. Freezing medium was

prepared by adding 10% (v/v) DMSO to normal cell culture medium. 1 × 106 cells

were centrifuged (167 × g, 5 min), the supernatant was discharged. The pellet was

resuspended in 1 ml freezing medium (4°C) and transferred into a cryo-vial. The

cryo-vial was placed in a freezing container filled with isopropanol and frozen at

-80°C with a cooling rate of 1°C per minute. The next morning, the cryo vials were

transferred into a liquid nitrogen container.

2.2.4.1 Harvesting of Cells:

1. Suspension Cells

After incubation suspension cell cultures were thoroughly mixed, the cell density

determined by coulter counter and the amount desired used in the assay.

2. Adherent Cells

In the case of adherent cells, the media was removed, cells washed twice with

generous amounts of PBS and detached with trypsin-EDTA (1 ml/25 cm2 adhered

cells). The trypsin digestion was stopped by adding the threefold amount of media.

Afterwards cells were separated by 3 – 5 times resuspension with a multipipette. The

cell density was determined by coulter counter.

2.2.5 Treatment of Cells for Testing

For toxicity testing of uremic toxins cells were seeded with a density of 2 × 105

cells/ml media (suspension cells; HL60, L5178Y) or 4 × 104 cells/cm2 (adherent cells;

LLC-PK1, CaCo-2). After letting the cells attach to the bottom of the flask or simply

adjust to the new media for 2 – 3 h the test substance was added. The concentration

of stock solutions for each test was chosen to result in a final solvent concentration of

0.1% (ethanol), 1% (DMSO) or 2% (ddH2O) in the media. These solvent

concentrations were known to cause no effect in the cell culture.

For the toxicity testing of eluates L5178Y cells were seeded with a density of

2 × 105 cells/ml media. After they had adjusted to the new media, eluates were

added to a final concentration of 2% (ethanol containing eluates) or 4% (ddH2O

eluates).

C Materials & Methods Toxicological Test

42

3 Toxicological Test

3.1 Frequent Test Substances

Frequent test substances are listed in Tab. C-6.

Test substance Manufacturer Solvent Stock solution

5-Aza-2’-deoxycytidine

Sigma-Aldrich, Taufkirchen, Germany, Cat. No: A3656

DMSO 250 µM

5-Aza-cytidine Sigma-Aldrich, Taufkirchen, Germany, Cat. No: A2385

DMSO 250 µM

Bisphenol A (BPA) Sigma-Aldrich, Taufkirchen, Germany, Cat. No: 239658

Ethanol 1 mM

DL-Homocysteine (Hcy)

Sigma-Aldrich, Taufkirchen, Germany, Cat. No: 53510

ddH2O 500 mM

H2O2 Sigma-Aldrich, Taufkirchen, Germany, Cat. No: 31642

ddH2O 50 mM

Homocysteine-Thiolactone (Hcy-T)

Sigma-Aldrich, Taufkirchen, Germany, Cat. No: H6503

ddH2O 500 mM

Leptin Sigma-Aldrich, Taufkirchen, Germany, Cat. No: L4146

DMSO 1 mg/ml

Methyl-Methane-sulfonate (MMS)

Sigma-Aldrich, Taufkirchen, Germany, Cat. No: M4016

DMSO 50 µg/ml

Mitomycin C (MMC) Sigma-Aldrich, Taufkirchen, German,

Cat. No: M4287 ddH2O 12.5 µg/ml

NaAsO2 Sigma-Aldrich, Taufkirchen, Germany, Cat. No: S7400

ddH2O 10 mM

Tab. C-6 Frequent test substances

Additional toxicity tests were performed with the serum of HD patients with

elevated MN frequency. The serum was obtained through another study (Treutlein, to

be submitted).

3.2 Cytotoxicity

3.2.1 Proliferation

3.2.1.1 Theoretical Background

One parameter of cytotoxicity which can be obtained fast and easily is cell

proliferation. A reduction of proliferation is an indicator for either direct cytotoxic effect

by induction of apoptosis or necrosis or indirectly by a diminished proliferation rate.


43

3.2.1.2 Procedure

After seeding of cells and addition of test substances, cells were incubated for

24 h to 48 h. Thereafter cells were harvested and the cell number determined by

coulter counter.

3.2.2 BrdU Incorporation Assay


The BrdU incorporation assay can be used to evaluate changes in the cell cycle -

e.g. cell cycle arrest - which can be caused by toxic substances. In this assay, BrdU -

an analogue of the DNA precursor thymidine - is incorporated into newly synthesized

DNA of cells entering and progressing through the S-phase (DNA synthesis) (Becton

2005). The incorporated BrdU can then be stained by an anti-BrdU antibody. The

combination with a total DNA staining by 7-amino-actinomycin (7-AAD) permits the

characterization of the cells in regard to their cell cycle position (G0/G1, S or G2/M

phase).

3.2.2.2 Materials and Buffers

• BrdU Flow Kit (BD Pharmingen, Heidelberg, Germany, Cat. No. 559619)

• Staining buffer: 3% FBS, 0.9% sodium acid in PBS

3.2.2.3 Procedure

1. Incubation of Cells with Test Substance:

Cells were seeded with a density of 2 × 105 cells/ml and the test substance

added. To ensure that the cells were in the exponential growth phase during the

assay, cell density was readjusted to 2 × 105 cells/ml, 10 h prior to the actual start of

the assay. The new test substance was added and the cells kept in the incubator (5%

CO2; 37°C) for the remaining incubation period.

2. Labelling of Cells with BrdU:

10 h later BrdU was added to the media to give a final concentration of 10 µM

BrdU. During BrdU incorporation cells were kept in an incubator (5% CO2; 37°C).

After 30 min incorporation time, cells were centrifuged (200×g) and washed with 1 ml


44

staining buffer per sample. During incorporation time kit components were diluted

according to the manual.

3. Fixation and Permeablisation of Cells:

After an additional centrifugation step, the supernatant was discharged and the

cells resuspended in 100 µl BD Cytofix/Cytoperm™/sample. The cells were

incubated on ice for 30 min. Next, cells were washed with 1 ml BD Perm/Wash

buffer™. This was followed by an additional permeabilisation step by incubation with

100 µl BD Cytoperm Plus buffer™ for 10 minutes on ice. After an additional washing

step cells were re-fixated for 5 minutes with 100 µl BD Cytofix/Cytoperm™/sample;

followed by another washing step.

4. Treatment of Cells with DNase:

Cells were treated with DNase (100 µl DNase/sample) to expose incorporated

BrdU. Next, cells were incubated in a 37°C water bath for 1 h.

5. Staining of BrdU with Fluorescent Antibodies:

After an additional washing step the BrdU was stained with 50 µl FITC labelled

Anit-BrdU-antibody (1:50 in PBS) for 20 min at room temperature. Surplus antibody

was removed by washing the cells with BD Perm/Wash buffer™.

6. Staining of total DNA:

The total DNA was stained by resuspending the cells in 20 µl 7-AAD solution

included in the kit. For analysis 1 ml staining buffer was added.

7. Flow Cytometric Analysis:

The flow cytometric analysis was performed at a run of less than 400 events/sec.

15,000 cells were analysed per sample. The cells were depicted in a dot blot forward

scatter (FSC, x-axis) versus side scatter (SSC, y-axis). The FSC is a parameter for

the size of the cells, the SSC for the inner granularity of cells. The cell population was

focused by adjustment of the forward scatter (FSC).

In a second dot plot the 7-AAD fluorescence (x-axis) was plotted versus FITC

anti-BrdU fluorescence (y-axis). 7-AAD fluorescence - representing the staining of the

whole DNA - was measured on channel FL3 (red fluorescence, 670 nm bandpass

filter). FITC anti-BrdU fluorescence – representing the staining of integrated BrdU into

newly synthesized DNA – was measured on channel FL1 (green fluorescence,


45

530 nm bandpass filter). In order to quantify the cell cycle positions region gates were

applied. This allowed the discrimination between subsets of cells that were apoptotic

(sub G1/G0) or resident in G0/G1, S or G2 + M phases of the cell cycle and had

recently synthesized DNA (Fig. C-1). The optimum settings were determined with the

untreated control cells.

Sub G0/G1-phase G0/G1-phase G2/M-phase S-phase

Fig. C-1 Exemplary dot plot of a quantitative cell cycle analysis of control cells, stained for

incorporated BrdU and total DNA levels

3.3 Genotoxicity

3.3.1 Comet Assay (Single-Cell Gel Test)


The comet-assay or single-cell gel test is a simple, sensitive and fast test for

studying DNA damage and DNA repair (Speit and Hartmann 1998). In this method a

small amount of cells are embedded into a thin layer of agarose on a microscope


46

slide. Subsequently the cells are lysed, electrophoresed and stained by a DNA-

intercalating dye. Cells with more DNA damage display a higher migration of

chromosomal DNA away from the nucleus (“head”) towards the anode, thereby

forming a “tail”. This results in the typical form of a comet (Fig. C-2).

a) untreated L5178Y cell (vehicle control)

b) L5178Y cell treated with 50 µM methyle-methane-sulfonate for 4 h

(positive control)

Fig. C-2 Exemplary pictures of cells in the comet assay

The alkaline version of this test – which was used in this PhD-thesis – was first

described by Singh and co-workers in 1988 (Singh, McCoy et al. 1988). This test is

used to monitor single-strand breaks and alkali-labile sites of the DNA. Several other

variations have been described which allow e.g. the detection of cross links (Pfuhler

and Wolf 1996).

The advantages of the comet assay are the easy and fast performance, the

sensitivity, the need for extremely small samples and applicability for nearly every

eukaryotic cell type. Therefore this test has become widely accepted for

pharmaceutical tests.

3.3.1.2 Buffer & Solutions:

• 1.5% Agarose (Agarose MEEO, Carl Roth GMBH, Karlsruhe, Germany, Cat.

No: 2268.2) in PBS

• 0.5% low melting point (LMP) agarose (Agarose Typ VII, Sigma-Aldrich,

Taufkirchen, Germany, Cat. No: A4018) in PBS

• Lysis buffer: 2.5 M NaCl, 100 mM EDTA, 10 mM TRIS, 1% w/v N-Lauroyl-

sacrosine sodium salt

• Lysis solution: 1% Triton, 10% DMSO in lysis buffer

• Electrophoresis buffer: 0.3 M NaOH, 1 mM EDTA


47

• Tris buffer: 0.4 M TRIS, pH 7.5

• Propidium iodide solution: 20 µg PI in double-distilled water (ddH2O)

3.3.1.3 Procedure:

1. Preparation of Slides and Buffers:

Superfrost slides were pre-coated with a layer of 1.5% agarose. Lysis solution

and electrophoresis buffer were prepared and stored at 4°C for at least 1 h prior to

use. The 0.5% LMP agarose was heated in the microwave oven and place in a 37°C

water bath.

2. Harvesting of Cells:

After incubation the period cells (HL60 or L5178Y) were harvested and the cell

density was adjusted to 0.8 - 1.2 × 106 cells/ml

3. Coating of Slides with Cells:

For each sample, two slides were prepared. Thereby 180 µl of the LMP-agarosis

solution were mixed with 20 µl cell suspension in a pre-warmed Eppendorf tube.

45 µl of this mixture were dropped on the pre-coated agarose slide and covered with

a cover slip. After the agarose had hardened the cover slip was removed.

4. Lysis:

The slides were transferred to a cuvette filled with lysis solution (4°C). Lysis was

performed at 4°C in the dark for 1 - 18 h.

5. Unwinding of DNA and Electrophoresis:

After lysis, slides were placed in a horizontal electrophoresis unit, filled with

electrophoresis buffer. The slides were left in the shaded unit for 20 min in order to

allow for unwinding of the DNA. Then electrophoresis at 25 V and 300 mA for 20 min

followed.

6. Neutralization and Staining:

After electrophoresis slides were neutralized in 0.4 M tris buffer for 5 - 10 min.

Slides were stained with 15 µl PI solution and covered with a cover slip. Slides were

stored at 4°C under humid conditions until analysis.


48

7. Analysis:

A fluorescence microscope (Nikon, Labphot 2 A/L, Nikon, Germany) equipped

with a camera (Cohn-High Performance, CDD Camera, Intas Sciences, Germany)

and Komet 5 software (Kinetic Imaging, UK) was used to analyse the slides. The

percentage of DNA in the tail of the comet was used to determine DNA damage. Two

slides per sample were evaluated by analysing 25 cells per slide.

3.3.2 Micronucleus Test


Micronuclei (MN) are DNA-containing structures which are formed during mitosis

and result from chromosomal breaks lacking centromers and/or whole chromosomes

incorrectly distributed during mitosis. At telophase a nuclear envelope forms around

these chromosomes or fragments, thereby forming nucleus-like structures, except

that they are smaller (see Fig. C-3).

Fig. C-3 Schematic formation of micronuclei

During mitosis chromosomal breaks or chromosomes lacking centromers are

incorrectly distributed. While the cell undergoes telophase, a nuclear envelope

forms around these chromosomes or fragments thereby forming nucleus like

structures - micronuclei

The MN represent a subgroup of all chromosomal aberrations, which makes the

MN frequency test a widely accepted method for in vitro and in vivo genotoxicity

investigation and human biomonitoring studies (Miller, Potter-Locher et al. 1998;

Fenech 2000; Kirkland, Henderson et al. 2005). In vitro MN can be induced by a

variety of genotoxic effects, for instance double strand breaks, oxidative stress,

inhibition of the spindle formation or even interference with the DNA methylation.


49

It is evident that MN can only be formed by dividing cells; therefore the assay can

not be used in non-dividing cell populations. Consequently the MN test was

advanced and the cytokinesis-block micronucleus assay developed (Fenech and

Morley 1985; Fenech and Morley 1985). In this assay cells undergoing nuclear

division are blocked from cytokinesis using the inhibitor of actin polymerisation

cytochalasin-B (Cyt B). Consequently the cells which have undergone one mitosis

can be distinguished from the rest by their binucleated appearance. Usually the

number of MN per 1000 binucleated cells is the determined.

The cytokinesis-block micronucleus assay can be also used to measure

additional reactions towards cytotoxic and genotoxic events, like: necrosis, apoptosis,

nucleoplasmatic bridges and cytostasis (Fig. C-4)

Fig. C-4 Fate of cells following exposure to cytotoxic/genotoxic agents

(modified after Fenech 2002).


50

3.3.2.2 Materials:

• Cyt B stock solution: 1 mg/ml cytochalasin B (Sigma-Aldrich, Taufkirchen,

Germany, Cat. No: C6762) in DMSO.

• Staining solutions:

Acridine orange stock solution: 0.1% w/v acridine orange in ddH2O

Working solution: 6.3% v/v acridine orange stock solution in Soerensen

buffer

• Soerensen buffer: 15 mM Na2HPO4 × 2 H2O; 15 mM KH2PO4 × H2O; pH 6.8

3.3.2.3 Procedure:

1. Treatment of Cells with Cytochalasin B (optional):

Cytochalasin B (Cyt B) treatment was conducted in case the test substance had

an effect on cell proliferation. After treatment of cells with the test substance 4 µg/ml

Cyt B were added to the media. After a 24 h incubation period, cells could be

harvested for cytospin preparation.

2. Preparation of Slides:

After the incubation period cells were harvested. Approximately 50,000 cells were

placed on microscopic slides by cytocentrifugation (5 min, 200 × g). In order to

ensure sufficient slides for analysis, 4 slides per treatment sample were produced.

After a brief quality control by light microscopy (magnification: 100 ×), slides were

fixed with ice-cold methanol (-20°C) for at least 2 h.

3. Staining:

Immediately prior to analysis cells were removed from the methanol containing

vial and transferred to a cuvette filled with acridine orange working solution. After 3 -

5 minutes staining, the residue acridine orange was removed by two subsequent

washing steps in Soerensen buffer for 5 min. Thereafter slides were covered with a

cover slip and placed in a dark, humid chamber.

4. Analysis of Cells (without Cyt B:)

Slides were analysed using a fluorescence microscope with an excitation

wavelength of 450 - 490 nm (magnification: 500 ×).


51

Two slides per sample were analysed by counting 1000 cells per slide. The value

obtained was MN/1000 cells. Depending on the problem additional parameters like

number of apoptotic cells or number of mitoses were scored simultaneously.

5. Analysis of Cells treated with Cyt B:


wavelength of 450 - 490 nm (magnification: 500 ×). Two slides per sample were

analysed by counting 1000 cells per slide. The following parameters were

determined:

• number of mononucleated cells

• number of binucleated cells

• number of polynucleated cells

• number of apoptotic cells

• number of cells undergoing mitosis

In order to determine the micronucleus frequency, 1000 binucleated cells were

counted and the number of MN per binucleated cells determined.

3.3.3 Determination of DNA-Cytosine Methylation by Flow

Cytometry

3.3.3.1 Theoretical Background:

DNA methylation is a chemical modification of the DNA by covalent addition of a

methyl group to the 5’carbon of cytosine within the context of the CpG nucleotide (s.

introduction). Changes in the overall DNA methylation can be analysed by a flow

cytometric analysis of DNA stained with an anti-5-methyl-cytosine antibody.

3.3.3.2 Materials

• Fixation buffer: 0.25% Paraformaldehyde (Sigma-Aldrich, Taufkirchen,

Germany, Cat. No: 158127) in PBS

• Wash buffer: 1% BSA, 0.2% Tween 20 in PBS

• 88% Methanol in PBS

• 2 N HCl (Carl Roth GmbH, Karlsruhe, Germany)

• Staining solution: 0.1% BSA in PBS


52

• Neutralizing buffer: 0.1 M Borate solution (Sigma-Aldrich, Taufkirchen,

Germany, Cat. No: A7906); pH adjusted to 8.5 with NaOH

• Stop solution: Wash buffer with 10% FBS

• Primary antibody: anti-5-methyl-cytosine (Calbiochem, San Diego, USA, Cat.

No: 16233 D3), stock solution: 1 mg/ml; final concentration: 2 µg/ml.

• Secondary antibody: FITC conjugated goat anti mouse antibody (Dianova,

GmbH, Hamburg, Germany, Cat. No: 115-095-003); stock solution: 1.5 mg/ml;

working concentration: 1:50 in wash buffer.

• Propidium iodide 1 mg/ml

3.3.3.3 Procedure:

1. Treatment of L5178Y Cells:

Given that detectable changes in DNA methylation can require more than one

cell cycle, cells were treated for 72 h or a minimum of 3 cell cycles. Every 24 h media

was changed, cell density adjusted to 2 × 105 cells/ml and new test substance added.

Cells incubated with the known methylation inhibitor 5-aza-cytidine served as positive

control.


After the incubation period 1 × 106 cells were transferred into falcon tubes. Cells

were centrifuged (250 × g, 5 min) and washed twice with PBS.

3. Fixation:

The pellet was resuspended with 500 µl fixation buffer and vortexed briefly. Cells

were incubated at 37°C for 10 minutes and additionally on ice for 10 minutes. During

gentle vortexing 4.5 ml ice cold methanol were added und the suspension was left at

-20°C for 15 minutes. Cells were centrifuged (250 × g; 5 min) washed twice with 2 ml

washing buffer per sample.

4. DNA Denaturation and Neutralization:

Cells were resuspended in 1.5 ml HCl and incubated in a water bath (37°C) for

25 min. To neutralize HCl 1.5 ml borate buffer were added. The mixture was kept at

room temperature for 10 min. Thereafter, cells were washed twice with 2 ml washing

buffer.


53

5. Staining:

Prior to staining cells were immersed in 2 ml stop solution (30 min; 37°C).

Thereafter, cells were centrifuged (250 g; 5 min) and washed with 2 ml washing

buffer. Cells were mixed gently with 300 µl primary antibody solution. The suspension

was incubated in a water bath (37°C) for 40 minutes. Subsequently, two washing

steps with 2.5 ml washing buffer followed. The cells were resuspended with 300 µl

secondary antibody solution and incubated in a water bath (37°C) for 40 min. After

two additional washing steps with 2.5 ml washing buffer, cells were resuspended with

staining solution.


Flow cytometric analyses were performed at a run of approximately

400 events/sec. 20,000 cells were analysed each sample. The cells were depicted in

a dot blot FSC (x-axis, cell size) versus SSC (y-axis, inner granularity). The cell

population was focused by adjustment of the FSC. The optimum settings were

determined with untreated control cells.

In a histogram the FITC fluorescence (x-axis) was plotted versus the number of

cells (y-axis). FITC anti-5’methyl-cytosine – representing the staining of DNA

methylation – was measured on channel FL1 (green fluorescence, 530 nm band

pass filter) (Fig. C-5). A shift of the peak to the left shows a decrease of bound anti-5-

methyl-cytosine antibody. This indicates a decrease of overall DNA methylation.


54

Fig. C-5 Exemplary histogram of a flow cytometric analysis of L5178Y cells, stained against

5’-methyl-cytosine. The solid filled graph matches cells not stained by antibody; the

non-filled graphs match cells stained by antibody.

3.3.4 Determination of DNA-Cytosine Methylation by LC-MS/MS


Another possibility to determine overall DNA methylation is by HPLC-MS/MS. In

this method the DNA of treated cells is isolated and digested to the

2’deoxyribonucleosides. The amount of 5-methyl-2’deoxycytidine (5-mdCyd) and

2’deoxyguanosine (dGuo) is analyzed by LC-MS/MS. The DNA cytosine methylation

is determined as the quotient 5-mdCyd/dGuo based on the assumption that the sum

of deoxycytidine and 5-mdCyd equals dGuo in genomic DNA. The DNA-Cytosine

methylation measurement by LC-MS/MS was performed by Andreas Brink (method

described in (Fink, Brink et al. 2007).

3.3.4.2 Materials:

• DNA isolation kit (Nucleobond AX, Macherey-Nagel, Dueren, Germany)

Anti-5’methyl-cytosine


55

• C18 HPLC column (Reprosil Pur ODS, 2.0 mm x 150 mm; 5 µm; Dr. Maisch,

Ammerbuch, Germany)

• Analyst 1.4.1 (API 3000, Applied Biosystems)

• 5-methyl-2’deoxycytidine standard solution (Sigma-Aldrich GmbH, Munich,

Germany)

• 2’deoxyguanosine standard solution (Sigma-Aldrich GmbH, Munich, Germany)

• Buffer A: 25 mM CH3CO2NH4 (Sigma-Aldrich GmbH, Munich, Germany, Cat.

No: A1542), 1 mM ZnCl2 in ddH2O; pH 5.1

• Buffer B: 100 mM NH4HCO3 (Sigma-Aldrich GmbH, Munich, Germany, Cat.

No: A6141), pH 8.0

• Buffer C: 1 M CH3CO2NH4 (Sigma-Aldrich GmbH, Munich, Germany, Cat. No:

A1542), 45 mM ZnCl2 in ddH2O; pH 5.1

• Buffer D: 1.5 M NH4HCO3 (Sigma-Aldrich GmbH, Munich, Germany, Cat. No:

A6141), pH 8.0

• Nuclease P1: (Sigma-Aldrich GmbH, Munich, Germany, Cat. No. N8630),

working nuclease: dissolved in buffer A to a final concentration of 1 unit/µl.

• Alkaline phosphatase: (Calbiochem, San Diego, California, USA, Cat. No.

524576); working solution: dissolved in buffer B to a final concentration of

200 units/ml

• Amicon Ultafree®-MC centrifugal units: (Millipore, Schwalbach, Germany, Cat.

No. UFC3LCC00)

3.3.4.3 Procedure:

1. Treatment of Cells:

Given that detectable changes of DNA methylation can require more than one

cell cycle, cells were treated for 72 h or at least 3 cells cycles. Every 24 h the media

was changed, cell density adjusted to 2 × 105 cells/ml and the new test substance

added.

2. DNA Isolation:

After incubation the DNA was isolated according to the DNA isolation kits

manual. In brief, 5 × 106 cells were washed twice with PBS and treated with the

buffer supplied to disrupt the cell membrane. Proteins were digested by addition of

proteinase K (20 mg/ml) followed by an incubation step (50°C, 60 min). After


56

equilibration the DNA was loaded onto a cartridge, washed three times and eluted

again. The DNA was precipitated over night at 4°C by the addition of isopropanol.

The resulting pellet was dissolved in 10 µl ddH2O.

3. Photometrical Analysis of DNA Quantity:

1 µl of dissolved DNA was diluted with 99 µl ddH2O and transferred to an acryl

cuvette. DNA content was determined by measuring the absorption at 260 nm.

Triplicates for each sample were prepared and measured twice. The DNA content

was calculated applying the following equation:

(Absorption260 × dilution factor × 50)/1000 = DNA µg/µl

Approximately 5 × 106 cells resulted in 5 to 20 µg of DNA.

The DNA concentration of the samples was adjusted to approximately 1 µg

DNA/µl.

4. DNA Hydrolysis:

DNA was digested to the 2’-deoxyribonucleosides by adding 1 µl of buffer C to

20 µl sample (≈ 20 µg DNA). In order to liberate the nucleotides from the DNA the

sample was incubated with 2 µl Nuclease P1 (0.1 units per µg DNA) for 120 min at

40°C. Then, 2 µl buffer D as well as 2 µl alkaline phosphatase (0.02 units/µg DNA)

were added to the sample to catalyze the hydrolysis of 5′-terminal phosphates of

DNA. The incubation was performed at 40°C for 60 min. Thereafter, the sample was

centrifuged (20 min; 7200 × rpm, 4°C) in a 5000 atomic mass units (amu) cut-off filter

tube to remove the enzymes.

5. LC-MS/MS Analysis:

Prior to analysis of 5-mdCyd and dGuo the samples were diluted with ddH2O

(1:100). 10 µl were injected on a C18 HPLC column Reprosil Pur ODS using an

Agilent 1100 autosampler and an Agilent 1100 HPLC-pump. The samples were

separated by gradient elution with water containing 0.1% formic acid (Solvent A) and

acetonitril (Solvent B) employing the following conditions: 10% B linear to 40% in

3 min, then linear to 100% within 2.5 min at a flow rate of 300 µl/min. The eluent was

analysed in the multiple reactions monitoring modus using a triple-stage quadruple

mass spectrometer equipped with an electrospray ionization source, controlled by


57

Analyst 1.4.1. The ion spray voltage was 3400 V, the source temperature 400°C. The

transitions monitored with a dwell time of 0.1 seconds each for 5-mdCyd and dGuo,

were m/z 242 → 126 and m/z 268 → 152. Declustering potentials were set to 16 and

20 V, focusing potentials to 130 and 200 V for 5-mdCyd and dGuo respectively. The

collision energies were set to 55 and 30 V for 5-mdCyd and dGuo, the collision cell

exit potentials were 10 and 15 V respectively.

Calibration curves were constructed using 5-mdCyd and dGuo standard

solutions. DNA cytosine methylation was determined as 5-mdCyd/dGuo using dGuo

as internal standard based on the assumption that the sum of dCyd and 5-mdCyd

equals dGuo in genomic DNA.

3.4 Oxidative Stress Measurement

3.4.1 Reactive Oxygen Species Measurement:


2`,7`-Dichlorofluorescein diacetate (DCFH-DA) can be used to measure

intracellular oxidant production (Robinson, Bruner et al. 1988). DCFH-DA is a non-

fluorescent, membrane permeable substance which is enzymatically hydrolysed to

the non-fluorescent DCFH by intracellular esterases. In the presence of ROS, DCFH

is easily oxidized to the highly fluorescent 2`,7`-dichlorofluorescein (DCF). The DCF

signal is analysed by flow cytometry, giving information about the amount of

oxidative stress in the cells.

3.4.1.2 Materials:

• 2`,7`-Dichlorofluorescein diacetate (Serva, Heidelberg, Germany, Cat.

No:19353) working solution: 20 mM DCFH-DA in DMSO

• 1% PBS-BSA: 1% BSA in PBS

• Propidium iodide solution: PI working solution: 100 µg/ml in ddH2O

3.4.1.3 Procedure:

1. Treatment with DCFH-DA:

15 min prior to incubation with the test substance, DCFH-DA was added to the

media at a final concentration of 10 µM.


58

2. Preparation of Cells for Analysis:

Cells were treated with the test substance and harvested after the desired

incubation period. Cells were washed twice with BSA-PBS and finally cells

resuspended in 1 ml BSA-PBS.

3. Staining with Propidium Idiodide:

10 µl PI working solution were added to the cells, resulting in a final

concentration of 1 µg/ml.


The flow cytometric analyses were performed at a run of 400 - 800 events/sec.

The fluorescence of 20,000 cells was measured. The cells were depicted in a dot blot

FSC (x-axis, cells size) versus SSC (y-axis, cell granularity). The cell population was

focused by adjustment of the FSC.

In a second dot plot the PI fluorescence (x-axis) was plotted versus the number

of cells. PI fluorescence was measured on channel FL3 (red fluorescence, bandpass

filter 670 nm). PI is not able to diffuse through membranes of living cells; therefore

living cells can be distinguished from dead cells through the lack of red fluorescence.

A gate was put around the living cells in the plot and a histogram showing their

DCF fluorescence (x-axis) vs. number of cells was constructed. DCF fluorescence

represents the amount of ROS within the cell. It was measured on channel FL1

(green fluorescence, bandpass filter 530 nm). A shift of the generated peak to the

right indicated an increase of ROS within the cells (Fig. C-6).


59

Fig. C-6 Exemplary oxidative stress measurement of HL60 cells using DCFH-DA as dye

Increased oxidative stress causes increased DCF fluorescence and therefore a

shift the peak to the left.

3.5 GSH/GSSG – Assay


Reduced glutathione (GSH) is one of the major antioxidants in nucleated cells. It

provides the reducing equivalent for the reduction of hydrogen peroxide and lipid

hydroperoxides. During this process GSH is oxidised to GSSG. GSSG is then

recycled to GSH by the glutathione reductase and NADPH.

This principle is used for the quantitative determination of total GSH. The method

was first described by Tietze (Tietze 1969). It employs 5`5-dithiobis-2-nitrobenzoic

acid (DTNB), which reacts with GSH resulting in a change of colour (Fig. C-7). This

change is proportional to the GSH and GSSG concentrations and can therefore be

used to quantify the total GSH concentration.


60

Fig. C-7 Reactions of the GSH-GSSG assay

GSH = reduced glutathione; GSSH = oxidized glutathione; DTNB = 5`5-dithiobis-2-nitrobenzoic acid; TNB = 2-Nitro-5-thiobenzoic acid; NADPH = Nicotinamide adenine dinucleotide phosphate (reduced); NADP

+ = Nicotinamide adenine dinucleotide phosphate (oxidized)

3.5.1.2 Materials:

• 0.1 mM Na2HPO4 buffer in ddH2O, pH 7.5

• 60 mM NADPH: (Sigma-Aldrich GmbH, Munich, Germany, Cat. No: N-1630) in

0.01 M NaOH

• 50 mM EDTA (Carl Roth GmbH, Karlsruhe, Germany, Cat. No: 8040.3) in

ddH2O

• 25 mM DTNB (Sigma-Aldrich GmbH, Munich, Germany, Cat. No: D-8130) in

ethanol

• 1% Sulfosalicylacid [m/m]

• Glutathione reductase (Roche, Mannheim, Germany, Cat. No: 10.105.768.

001)

• 10 mM reduced glutathione (Sigma-Aldrich GmbH, Munich, Germany, Cat. No:

G-4251)

• Reductase solution (for 10 measurements):

1.5 ml 100 mM phosphate buffer

30 µl 50 mM EDTA

15 µl 60 mM NADPH

8 µl reductase

1447 µl water

2 TNB

DTNB

GSSG

2 GSH

NADPH

NADP+


61

3.5.1.3 Procedure:

1. Sample Preparation:

1.5 × 106 cells were centrifuged (200 × g, 5 min, 4°C) and washed twice with cold

PBS. The pellet was resuspended in 400 µl sulfosalicylacid in order to disrupt the cell

membrane. Cells were left on ice for 15 minutes and centrifuged (5000 × g, 3 min,

4°C). The pellet was discargded; the cell extract used for measurement.

2. Photometric Measurement:

Prior to sample measurement a GSH calibration curve was established using 8

calibration points (250 µM, 500 µM, 750 µM, 1000 µM, 1250 µM, 1500 µM, 1750 µM,

2000 µM). For the sample measurement 20 µl cell extract were added to 260 µl

phosphate buffer. Then 20 µl DTNB and 300 µl reductase solution were added. The

kinetic was measured at 410 nm for 90 sec.

3. Data Analysis:

The slope of the kinetic was calculated using Microsoft Excel. The actual GSH

concentration was determined by comparison to the calibration curve.

3.6 Apoptosis

Apoptosis - the process of cell suicide – is an active process which can be

triggered by a multitude of external and internal signals, e.g. toxic cell injury. In

contrast to necrosis, apoptosis follows orderly steps. After the stimulus a signalling

cascade is set off. One of the first consequences is the destruction of the

cytoskeleton, which leads to shrinking and rounding of the cells. Furthermore the

chromatin undergoes condensation into compact patches against the nuclear

envelope, followed by DNA fragmentation. Moreover, the cell membrane forms

irregular buds and releases apoptotic bodies.

3.6.1 Bisbenzimide Staining:


One important feature of cells undergoing the apoptotic process is the irreversible

condensation of chromatin, followed by the fragmentation of the nucleus. These


62

changes can be analysed microscopically after staining of the total DNA with DNA-

binding dyes like bisbenzimide (Hoechst 33342), (Fig. C-8).

a) Apoptotic cell stained with

Hoechst 33342 (× 1000)

b) Untreated LLC-PK1 stained with

Hoechst 33342 (× 1000)

Fig. C-8 L5178Y cells stained with the Hoechst 33342 dye

3.6.1.2 Materials:

• Bisbenzimide (Hoechst 33342): Working solution: 50 µM in ddH2O

• Mounting media: Vectashield® Mounting Medium (Linaris, Wertheim,

Germany, Cat No: H-1000)

3.6.1.3 Procedure:

1. Preparation of Slides:

After harvesting, approximately 50,000 cells were placed on microscopic slides

by cytocentrifugation (5 min, 200 × g). In order to ensure sufficient slides for analysis,

4 slides per treatment sample were produced. After a brief quality control by light

microscopy (magnification 100 ×) slides were fixed in ice cold methanol (-20°C) for at

least 2 h.

2. Staining:

Prior to analysis, cells were removed from methanol and transferred to a cuvette

filled with bisbenzimide working solution. After 3 min staining, the residue

bisbenzimide was removed by two subsequent washing steps with PBS for

5 minutes. A coverslip was placed on the slide with one drop of mounting media.

Prepared slides were stored in a dark, humid chamber.


63

3. Analysis of Cells


wavelength of 330 - 380 nm. Two slides of each sample were analysed by counting

1000 cells per slide. The value obtained was number of apoptoses/1000 cells.

3.6.2 Annexin V Staining and FACS Analysis


During early stages of apoptosis phosphatidylserine from the inner layer of the

plasma membrane is translocated to the external surface of the cell. This

phosphatidylserine can be labelled with the fluorescence-conjugated binding protein

Annexin-V-Fluos. Since necrotic cells also expose phosphatidylserine due to the loss

of membrane integrity, apoptotic cells have to be distinguished from necrotic ones by

the simultaneous staining with PI. PI is a red-fluorescent molecule that stains nucleic

acids. PI is membrane-impermeant, allowing discrimination between vital and vital

but apoptotic cells on the on hand and necrotic cells on the other hand.

3.6.2.2 Materials:

• Annexin-V-Fluos: (Roche, Mannheim, Germany, Cat. No. 1828681)

• AnnexinV/PI- staining solution: 20 µl Annexin-V-Fluos, 20 µl PI solution, 960 µl

1x binding buffer

• Binding buffer (10×): 0.1 M HEPES (pH 7.4)

140 mM NaCl

25 mM CaCl2

• Propidum iodide: 1mg/ml

3.6.2.3 Procedure:


Cells were resuspended and the cell number determined by coulter counter.

1 × 106 cells were transferred into a falcon tube, centrifuged (5 min, 200 × g) and

washed once with 1× binding buffer.


64

2. Staining:

Cells were centrifuged (5 min, 200 × g) again and resuspended in 200 µl

Annexin-V/ PI staining solution. After 20 minutes staining, 900 µl binding buffer were

added to the cells.


The flow cytometric analysis was performed at a run of 400 - 800 events/sec. The

cells were depicted in a dot blot FSC (x-axis, cell size) versus SSC (y-axis; cell

granularity). The cell population was focused by adjustment of the FSC.

In a second dot plot Annexin-V-Fluos fluorescence (x-axis) was plotted versus PI

fluorescence (y-axis). Annexin-V-Fluos was measured on channel FL1 (green

fluorescence; 530 nm bandpass filter). PI fluorescence was measured on channel

FL3 (red fluorescence, 670 nm bandpass filter). The fluorescence of 20,000 cells was

acquired and the events gated into 4 quadrants (Fig. C-9).

Kontrolle 1.001

100 101 102 103 104

Annexin V Fluos

a)

pos Kt. NaAsO2.007

100 101 102 103 104

Annexin V Fluos

b)

Fig. C-9 Annexin-V/PI staining of L5178Y cells.

3.7 Test for Estrogenic Activity: E-Screen

3.7.1 Theoretical Background

Several methods for in vivo or in vitro analysis of the estrogenic activity exist.

One of the most popular in vitro tests is the so called E-screen. In this methodical

approach the estrogenic activity of a substance is tested by the proliferation of the

estrogen sensitive cell line MCF-7. These human breast cancer cells carry an

estrogen receptor and respond to xenoestrogens by increased proliferation. In order

to perform this test properly it is important to avoid any hormonal activity in the

Control Positive Control

vital

cells

early

apoptosis

necr

osis

late

apoptosis

+ late

necrosis


65

media. Therefore the standard FBS in media is replaced by a charcoal /dextran

treated FBS, which is free of hormonal activity.

3.7.2 Material

• 0.02% EDTA Solution (Sigma-Aldrich GmbH, Taufkirchen, Germany, Cat. No:

E8008)

• Charcoal/Dextran treated FBS (HyClone, Logan, USA, Cat. No: SH30068.02)

(heat inactivated at 56°C for 30 minutes)

• Reduced media: Same media as for cell culture but the 10% FBS was

replaced by 5% FBS-DCC.

3.7.3 Procedure

1. Culture of Cells for the E-Screen

MCF-7 cells were cultured like any other adherent cell line, the only difference

being that the detachment of the cells occurred by 0.02% EDTA solution instead of

Trypsin-EDTA. This procedure is gentler to the cells and avoids accidental

destruction of the estrogen receptor on the cell surface.

2. Preparation of the E-Screens

MCF-7 cells were harvested using 0.02% EDTA solution. 12,000 cells/cm2 were

seeded in 25 cm2 cell culture flasks. The flasks were placed in an incubator (37°C,

5% CO2) for 24 h.

After 24 h the media was replaced by fresh reduced media and the test

substances added. Three replicates of each sample were produced. After 72 h the

media was changed and fresh test substance added. The cells remained in the

incubator for further 72 h.


Cells were washed twice with 5 ml with PBS and detached with 2 ml Trypsin-

EDTA per flask. The trypsin stayed on the cells for up to 10 min to allow an as good

detachment of the cell-cell as of the cell-matrix contacts. The trypsin was stopped by

addition of 3 ml media. To ensure separation cells were resuspended with a

dispenser tip.


66

4. Analysis:

Cells were counted by coulter counter. The values obtained were normalized to

the control (100%). The percental increase of growth resembles the estrogenic

activity of the substances.

3.8 Generation of Advanced Glycation End Products:


AGEs are a heterogenous group of compounds which are formed by non-

enzymatic reactions between reducing sugars and free amino groups of proteins,

followed by subsequent reactions. As it is difficult to test and characterize a

heterogenous group of molecules, two model AGEs were prepared:

carboxy(methyl)lysine-modified bovine serum albumin (CML-BSA) and

methylglyoxal-modified BSA (MGO-BSA).

3.8.1.1 MGO-BSA Production

Material

• 0.1 M Na2HPO4 buffer (Merck, Darmstadt, Germany, Cat. No: 567547)

• 40% Methylglyoxal (Sigma-Aldrich, Taufkirchen, Germany, Cat. No: M0252)

• BSA

Procedure

1. Production of MGO-Matrix:

2.65 g BSA were dissolved in 40 ml 0.1 M Na2HPO4 buffer. 10 ml of this solution

were kept as control. The remaining 30 ml were mixed with 633 µl methylglyoxal. The

solution and the control were filtered sterile.

2. Incubation:

The solutions were incubated for 7 days at 37°C.


67

3. Dialysis:

After incubation the solution as well as the control was transferred into dialysis

tubing membranes. MGO-BSA was now purified by dialysis against ddH2O for 72 h.

During dialysis ddH2O was changed several times (30 l in total).

4. Lyophilisation:

After dialysis the solutions were frozen at – 80°C and freeze-dried at - 58°C for

48 - 72 h.

5. Preparation of MGO Stock Solution:

Immediately prior to the experiment MGO was dissolved in PBS [c = 100 mg/ml].

3.8.1.2 CML-BSA Production

Materials

• 0.2 M Na2HPO4 buffer (Merck, Darmstadt, Germany, Cat. No: 567547)

• 10 M NaOH

• 1.5 M glyoxylic acid in Na2HPO4 buffer

• NaCNBH3

Procedure

1. Production of CML-Matrix:

2.65 g BSA were dissolved in 40 ml 0.1 M Na2PO4 buffer. 10 ml of this solution

were kept as control. The remaining 30 ml were mixed with 1 ml glyoxylic acid. The

solution was stirred at RT for 2 h. After adjustment of the pH to 7.4 (with 10 M

NaOH), 0.238 g NaCNBH3 were added. The solution and the control were filtered

sterilely.

2. Incubation:

The flasks were incubated at room temperature for 2 - 3 days.

C Materials & Methods Extraction of Eluates from Various Dialysers

68

3. Dialysis:

After incubation the solution as well as the control was transferred into tubular

dialysis membranes. CML-BSA was now purified by dialysis against ddH2O for 72 h.

During dialysis, ddH2O was changed several times (30 l in total).

4. Lyophilisation:

After dialysis, the solutions were frozen at – 80°C and freeze-dried at - 58°C for

48 - 72 h.

5. Preparation of CML Stock Solution:

Immediately prior to the experiment CML was dissolved in PBS [c = 100 mg/ml].

4 Extraction of Eluates from Various Dialysers

4.1 Conditions of Elution

The conditions of elution should resemble the real-life dialysis modalities as

closely as possible. Therefore the elution was performed in an incubator at 37°C ±

1°C, the temperature at which dialysis is performed. The eluting media of choice

should feature three characteristics: they should have similar extraction properties

like blood, they should not interfere with HPLC-MS/MS analysis and it should be

possible to use them in toxicity tests without complicated reconditioning, which could

interfere with the tests. Therefore barely volatile (e.g. oil) or saline solutions (e.g.

artificial serum) could not be used. For simplified analysis ddH2O was used.

Additionally a 17.2% ethanol (EtOH) solution was used because its extraction

properties towards BPA are similar to bovine serum (Haishima, Hayashi et al. 2001).

In order to prevent any contamination with ubiquitous existing substances like

phthalates, only glass equipment was used. All equipment was sterilized for 8 h at

240°C.


69

4.1.1 Materials:

• Eluents:

• HPLC grade water in glass bottles (Carl Roth GmbH, Karlsruhe,

Germany)

• 17.2% ethanol: HPLC grade ethanol in glass bottles (Carl Roth

GmbH, Karlsruhe, Germany) diluted with HPLC grade water

• Dialysers (Tab. C-7):

• FX60, F60S, FX80, HF80S (Fresenius Medical Care, Bad

Homburg, Germany)

• 170 H (Gambro, Hechingen, Germany)

Dialysers Lot-Number Potting material

Sealing ring

Housing Membranes Surface

area

FX60 LDV15104

NBV12101 Polyurethane Silicone Polypropylene

Helixone® (Polysulfone –PVP blend)

1.4 m2

F60S LCB29101 Polyurethane Silicone Polycarbonate Fresenius

Polysulfone®(Polysulfone –PVP blend)

1.3 m2

FX80 NCX01130

NGV3021 Polyurethane Silicone Polypropylene

Helixone (Polysulfone –PVP blend)

1.8 m2

HF80S NAK24160

NEK11140 Polyurethane Silicone Polycarbonate

Fresenius Polysulfone®(Polysulfone

–PVP blend) 1.8 m

2

170 H S-4402-H-01 Polyurethane Silicone Polycarbonate Polyamide S

(Polyamid/Polysulfone blend)

1.7 m2

Tab. C-7: Dialysers and their properties

• Tubing:

• PVC (Sis-Ter SpA, Palazzo Pignano, Italy)


70

4.1.2 Procedure:

1. Assembly of the Dialysis Equipment

In order to elute all leaching substances from the dialyser, the blood and the

dialysate compartment were connected by 110 cm standard PVC tubes. The eluent

was poured into the dialyser using only sterilized glass equipment. The approximate

volume of eluent filled in the dialysers is given in Tab. C-8. Eventually, the tubes of

the filled dialyser were connected to a peristaltic pump (Fig. C-10).

Fig. C-10 Assembly of the dialysis equipment

2. Dialysis:

Elution was performed in an incubator at 37 ± 1°C with a flow rate of 230 ml

eluate/min for 4 h or 24 h, respectively. The 4 h time period was equivalent to the real

time of clinical dialysis, the 24 h period resembled the worst case scenario. At least 3

independent eluates with new dialysers each time were obtained at each elution

condition.

3. Processing of the Eluates:

After the desired elution time the extraction was stopped. Only 51 - 64%

(depending on the dialyser type) of the fluid used could be regained (Tab. C-8). This

was due to absorption of eluate in the hollow fibres system.

This fluid was poured into 250 or 500 ml flasks. Thereafter the ethanol was

removed by rotary evaporation at 38 - 40°C. The remaining eluate was frozen at

-80°C and lyophilised at - 58°C for 2 - 4 days. The solid substance obtained was

dissolved in 2 - 4 ml eluate, depending on the ease of solubility. Dissolving in 2 ml

eluate was preferred, because this resulted in a higher concentration of leaching

C Materials & Methods HPLC-MS/MS Analysis

71

substances and therefore finally in a higher concentration in the cell culture test

systems. Four ml eluent were only used in case solubility in 2 ml could not be

achieved.

To obtain any substance potentially left in the flask, the flask was washed with

additional eluent. The analyses described below were performed with the first eluate

and the second eluate.

In order to determine the quantity of contaminations originating from the

treatment of the eluates and not from the dialyser, 300 ml of ddH2O or 17.2% ethanol

were filled into flasks and treated like the eluates. They served as control.

Dialysers Eluent utilised

Eluate obtained

Percentage regained

FX60 ≈ 230 ml ≈ 125 ml ≈ 54%

F60S ≈ 315 ml ≈ 200 ml ≈ 64%

FX80 ≈ 285 ml ≈ 145 ml ≈ 51%

HF80S ≈ 435 ml ≈ 280 ml ≈ 64%

170 H ≈ 410 ml ≈ 250 ml ≈ 61%

Tab. C-8 Utilized and obtained eluate from different dialysers

5 HPLC-MS/MS Analysis

5.1 Full Range Scan


In order to get a general idea of substances detectable in the eluates, a full range

scan of dialyser FX60 and F60S eluates was performed. In this method ions are not

filtered so every mass can be detected.

5.1.2 Materials

• Column: Phenomenex Synergi 4a Hydro RP (4.0-µm, 150 x 2,0 mm,

Phenomenex, USA)

• ddH2O: HPLC gradient grade quality (Carl Roth GmbH& Co, Karlsruhe,

Germany)

• Acetonitrile: HPLC gradient grade quality (Carl Roth GmbH& Co, Karlsruhe,

Germany)

• Analysis software: Analyst 1.4.1 software (Applied Biosystems, Darmstadt,

Germany)


72

5.1.3 Procedure

Samples (10 µl) were separated by a Phenomenex Synergi 4a Hydro RP column

using Agilent 1100 autosampler and an Agilent 1100 HPLC pump. Gradient elution

with water (solvent A) and acetonitrile (solvent B) was applied with the following

conditions: 1 min 90% B, 10% A; for 14 minutes a linear increase up to 100% A;

5 min 100% A; for 2 min decrease of the gradient up to 10% A and 90% B. The

gradient was maintained for a further 3 min. By using this gradient, hydrophilic as well

as hydrophobic substances could be eluted from the column and detected later on.

The flow rate was 300 µl/min. The experiments were performed on a linear ion trap

mass spectrometer equipped with a TurbolonSpray source connected to the HPLC

system. To record spectral data, a vaporizer temperature of 450°C and a

TurblonSpray voltage of -4.5 kV in the in the positive ion mode were applied.

5.2 BPA Analysis


One of the molecules expected in the sample matrix was BPA. The analysis was

performed according to Völkel et al by L-MS/MS using API 3000 (Volkel, Bittner et al.

2005).

5.2.2 Materials

• Column: Reprosil-Pur ODS-3 (5 µM, 150 × 4.6 mm, Maisch, Ammerbuch,

Germany)

• ddH2O: HPLC gradient grade quality (Carl Roth GmbH& Co, Karlsruhe,

Germany)


Germany)

• Internal standard: d16-BPA (generated and provided by Dr. Völkel, Institute of

Toxicology, Würzburg, Germany), (Volkel, Colnot et al. 2002)

• Analysis software: Analyst 1.4.1 software (Applied Biosystems, Darmstadt,

Germany)


73

5.2.3 Procedure

Prior to analysis, eluate samples were spiked with 20 ng/ml internal standard d16-

BPA. Samples (10 µl) were separated by a Reprosil-Pur ODS-3 column using an

Agilent 1100 autosampler and an Agilent 1100 HPLC pump. Gradient separation was

performed with water (solvent A) and acetonitirile (solvent B) as solvents: 40% A /

60% B for 2 min, followed by a linear gradient to 20% A and 80% B within the next

20 min; 80% B and 20% A for a further 2 min. To record spectral data, a vaporizer

temperature 450°C and a TurbolonSpray voltage of - 4.5 kV in the negative ion mode

were applied. The declustering potential was set to - 40 V and N2 was used as

collision gas. For analysis of the BPA content the MS/MS transitions m/z 227/212

(BPA quantifier) and m/z 403.2 - 113.1 (d16 BPA quantifier) were evaluated. The

enhanced resolution was performed at a scan rate of 250 amu/s and a fill time of

50 s. The declustering potential was set to -40 V.

Quantification of BPA was based on a calibration curve using 7 data points (0,

3.1 ng/ml, 6.2 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml), with R2 = 0.997.

The total quantity of eluted BPA was determined by summation of BPA in eluate 1

and 2. The limit of detection (d.l.) was ca.0.57 ng/ml (signal to noise 3). The limit of

quantification was ca. 3.4 ng/ml.

5.3 DEHP Analysis

5.3.1 Theoretical Background:

Several studies reported an elevation of di(2-ethylhexyl)phthalate (DEHP) levels

in the serum of ESRD patients after dialysis. Therefore the eluate content of DEHP

was analysed in vitro. DEHP analyses of eluates from extracorporal circuits were

performed.

5.3.2 Materials:

• Column: Luna Phenyl-Hexyl column (3 µm, 150 × 4.6 mm, Phenomenex, USA)


Germany)

• Formic acid (first grade, Sigma-Aldrich, Taufkirchen, Germany, Cat. No. 06440)

• Internal standard: Bis-(2-ethylhexyl)-phthalate PESANAL (Sigma-Aldrich,

Taufkirchen, Germany, Cat. No: 36735)


74

5.3.3 Procedure:

Before the analysis samples were diluted with acetonitrile containing 0.1% formic

acid (1:9) additionally 100 pg/ml internal standard was added. The internal standard

as well as the DEHP for the calibration curve was dissolved in acetonitrile containing

0.1% formic acid.

The diluted sample (10 µl) was separated by a Luna Phenyl-Hexyl column The

analysis was carried out isocratically with 98% acetonitrile (containing 0.1% formic

acid) and 2% of 0.1% formic acid. For analysis of the DEHP content the MS/MS

transitions m/z 391.2/57.1 were evaluated.

The calibration curve was calculated from 5 data points (10, 50, 100, 500,

1000 ng/ml) (R<0.99). The limit of detection (d.l.) was ca 20 ng/ml (signal to noise 3).

The limit of quantification was between ca 70 ng/ml (signal to noise 10).

D ResultsSubstances Extracted from Blood Circuits Containing Dialysers and Tubings

75

D Results

1 Substances Extracted from Blood Circuits Containing

Dialysers and Tubings

The eluates obtained from various dialysers were concentrated by freeze-drying.

After this procedure a white, uncongested substance remained (Fig. D-1). The

amount of substance depended primarily on the dialyser type and on the eluent.

Generally, 17.2% ethanol eluates contained more leaching substances than ddH2O

eluates.

Fig. D-1 Lyophilised eluate (F60S, 17.2% EtOH, 24 h) in a 250 ml flask

When dissolving the extracted substance in ≈ 4 ml pure eluent a viscous,

yellowish eluate liquid emerged from the 17.2% ethanol eluates. The dissolving of

ddH2O eluates resulted in clear, less viscous liquids. Those liquids were used for the

analysis and testing described below.

2 HPLC-MS/MS Analysis of Eluates

2.1 Total Ion Scan

In order to gather a general idea of substances present in the concentrated

eluates, total ion scans of 5 eluates were performed. Those eluates were obtained by

4 or 24 h extraction from FX60 dialysers using ddH2O or 17.2% EtOH as eluent.

Those total ions scans were compared to the ones of pure eluents which served as

control.

D Results HPLC-MS/MS Analysis of Eluates

76

An exemplary figure of a chromatogram is shown in Fig. D-2. The HPLC-grade

ddH2O is depicted by a red line and an exemplary eluate by a blue line. When looking

at these graphs it is apparent that a variety of ions was detectable even in purest

water. After water was pumped through a FX60 dialyser for 4 h, there were distinct

changes in the ion spectra (blue line). Especially palpable are the 2 additional peaks

at time point 1.18 and 1.71. However, the mass to charge values (m/z) of each peak

were extremely multifarious and could not be attributed to a single substance. One

exemplary mass spectrum is given in Fig. D-3. It shows all m/z values which are

derived from one single peak (elution time 1.18 min) of the full range scan shown in

Fig. D-2.

The variety of ions detectable by the total ion scan impeded the qualification of

single substances. Consequently, further analysis had to be focused on specific

substances, which could be expected in the eluates.

Fig. D-2 Chromatograms of full range scans of ddH2O (red) und an exemplary eluate (FX60,

ddH2O, 4 h; blue). The flow of eluent through the assembled unit caused distinct

changes in the chromatograph pattern.

Time [min]

Inte

nsity [

cp

s]

Mass Spectrum

see Fig. D-3


77

Fig. D-3 Exemplary mass spectrum of an eluate (FX60, ddH2O, 4 h) at time 1.18 min

2.2 Di(2-ethylhexyl)phthalate Analysis

One of the substances expected to be found in the assembled circuit was DEHP.

The DEHP content of eluates obtained from blood circuits containing PVC tubing was

analysed by HPLC-MS/MS. As the dialysers used (FX60 and F80S) did not contain

DEHP, any detected DEHP would have been leaching from the connecting tubings.

An exemplary chromatogram and corresponding mass spectrum of the DEHP

analysis is shown in Fig. D-4 and Fig. D-5. While the internal standard was verifiable

in each analysis, the DEHP concentration of the eluates did not reach the limit of

detection (20 pg/ml) (Tab. D-1). Therefore the amounts of DEHP leaching from PVC

tubings can be regarded as minimal and additional experiments using polyolefine

tubing (DEHP free) were abandoned.

m/z amu

Inte

nsity [

cp

s]


78

Fig. D-4 Chromatogram overlay of an exemplary eluate (F60S, 17.2% EtOH, 24 h) (red) and

100 pg/µl DEHP standard (blue).

Fig. D-5 Mass spectrum of 100 pg/µl DEHP standard

Dialyser Tubing Eluent Elution period DEHP

concentration per dialyser

Number of independent repetitions

4 h < d.l. 4 ddH2O

24 h < d.l. 6

4 h < d.l. 4 F60S PVC

17.2 % EtOH 24 h < d.l. 4

4 h < d.l. 2 ddH2O

24 h < d.l. 4 FX60 PVC

17.2 % EtOH 24 h < d.l. 4

Tab. D-1 Elution modalities for DEHP measurement and results;

d.l. = detection limit

Time [min]

DEHP peak

m/z [amu]

Inte

nsity [

cp

s]

Inte

nsity [

cp

s]


79

2.3 Bisphenol A Analysis

The BPA content of eluates obtained from 5 different dialysers under various

elution conditions was determined by HPLC-MS/MS analysis. An exemplary

chromatogram of BPA analysis and the corresponding mass spectrum is shown in

Fig. D-6 and Fig. D-7.

Fig. D-6 Chromatogram of an exemplary eluate (F60S, 24 h, 17.2% ethanol)

Fig. D-7 Mass spectrum of bisphenol A

BPA could be detected in each of the eluates. Generally, more BPA was

detected in 17.2% EtOH eluates than in water eluates. The content ranged from

33.4 ng/dialyser (170H) to 2,321 ng/dialyser (FX80) when 17.2% ethanol was used

as eluent and from 3.9 ng/dialyser (170H) to 44.8 ng/dialyser (F60S) when ddH2O

was used. Moreover the type of dialyser, the batches of the same dialyser (produced

from different batches of polysolufone granulate) and the size of the membrane

surface influenced the amount of leaching BPA. Most BPA could be extracted from

FX80 dialysers and the least amount from 170H and FX60 dialysers. In most cases

there was also more BPA detectable in the 24 h eluates than in the 4 h eluates. The

quantity of BPA determined in the eluates is summarized in Tab. D-2.

Time

m/z [amu]

BPA peak


80

Dialyser Eluent Time [c] BPA

[ng/dialyser]

S.D.

[ng/dialyser]

4 h 35.9 5.2 ddH2O

24 h 39.8 3.9 FX60

Lot: LDV15104 17.2% EtOH 24 h 49.6 1.5

ddH2O 4 h 18.4 1.8

4 h 405.7 164.0 FX60

Lot: NBV12101 17.2% EtOH 24 h 482.7 89

4 h 38.0 3.6 ddH2O

24 h 44.8 4.9

4 h 66.7 58.6

F60S

Lot: LCB29101 17.2% EtOH

24 h 89.3 13.4

4 h 38.5 5.2 ddH2O

24 h 37.2 7.2

4 h 1839.8 506.7

FX80

Lot: NCX01130 17.2% EtOH

24 h 2321.4 1074.9

4 h 610.3 124.8 FX80

Lot: NGV3021 17.2% EtOH

24 h 681.6 69.8

4 h 12.5 3.9 HF80S

Lot: NAK24160 ddH2O

24 h 17.7 1.9

4 h 480.5 22.2 HF80S

Lot: NEK11140 17.2% EtOH

24 h 719.8 19.3

4 h 3.9 0.11 ddH2O

24 h 4.4 0.7

4 h 34 4.4

170H

Lot:S-4402-H-01 17.2% EtOH

24 h 33.4 11.9

Tab. D-2 Amount of BPA detected in different eluates. Values are given as the mean of measurement of at least 3 eluates.

In order to estimate the whole amount of BPA leaching from the dialysers it has

to be considered that only 51% - 64% of the fluid filled into the dialyser could be

regained. Most of the residual fluid remained in the hollow fibres of the dialysers due

to capillary forces. Additionally a small part of the fluid remained in the dialysate

compartment and could not be regained.

Presuming that BPA was uniformly dissolved in the whole fluid the total amount

of leaching BPA was estimated (Tab. D-3). The maximum of leaching BPA was

estimated to be 4.3 µg/dialyser.

D Results Cytotoxicity Testing

81

Dialyser Eluent Time Estimated amount of leaching

BPA/dialyser [ng]

4 h 66.5 ddH2O

24 h 73.7 FX60

Lot: LDV15104 17.2% EtOH 24 h 91.9

ddH2O 4 h 34.1

4 h 751.3 FX60

Lot: NBV12101 17.2% EtOH 24 h 893.9

4 h 59.4 ddH2O

24 h 70

4 h 104.2

F60S

Lot: LCB29101 17.2% EtOH

24 h 139.5

4 h 71.3 ddH2O

24 h 68.9

4 h 3407

FX80

Lot: NCX01130 17.2% EtOH

24 h 4298.9

4 h 1130.2 FX80

Lot: NGV3021 17.2% EtOH

24 h 1262.2

4 h 18.7 HF80S

Lot: NAK24160 ddH2O

24 h 25.7

4 h 717.2 HF80S

Lot: NEK11140 17.2% EtOH

24 h 1074.3

4 h 6.4 ddH2O

24 h 7.2

4 h 55.7

170H

Lot:S-4402-H-01 17.2% EtOH

24 h 54.8

Tab. D-3 Estimated amount of leaching BPA per dialyser

BPA could even be detected in pure eluent. This is probably due the ubiquitous

presence of BPA, which can result in incidental contamination of the eluent - either

during the manufacturing or bottling or during the subsequent handling process.

However, even though the BPA concentration was above the limit of detection

(0.56 ng/ml), it was below the limit of quantification (3.42 ng/ml). The amount of BPA

leaching from standard PVC tubes (110 cm length) was also above the limit of

detection but below the limit of quantification. Therefore the contribution of the tubing

system to the total BPA content of our eluates is negligible.

3 Cytotoxicity Testing

In order to evaluate the cytotoxicity of eluates several test have been performed.

Generally, 200 µl of the concentrated ddH2O eluates or 100 µl of the concentrated

17.2% EtOH eluates were added to 5 ml cell culture media, leading to a 25 or 50-fold


82

dilution of the concentrated eluate. Due to the varying amounts of eluents obtained

from the different dialysers, this led to the following concentration in media compared

to the pure eluate (Tab. D-4):

Dialyser Amount of eluate

obtained

Amount of concentrated

eluate

Concentration factor

Concentration factor of eluate in culture media compared to

unprocessed eluate

FX60 125 ml ≈ 4 ml 31.25 1.25 (ddH2O) / 0.625 (EtOH)

FX60S 200 ml ≈ 4 ml 50 2 (ddH2O) / 1 (EtOH)

FX80 145 ml ≈ 4 ml 36.25 1.45 (ddH2O) / 0.725 (EtOH)

HF80S 280 ml ≈ 4 ml 70 2.8 (ddH2O) / 1.4 (EtOH)

170H 250 ml ≈ 4 ml 62.5 2.5 (ddH2O) / 1.25 (EtOH)

Tab. D-4 Eluate concentrations in cell culture

Assuming a blood volume of 5 l the concentration of leaching substances in

blood was 25 times (ddH2O eluates) or 50 times (17.2% EtOH) lower than the ones

reached in cell culture.

3.1 Cell Proliferation

First the simplest parameter of cytotoxicity – changes in cell proliferation – was

assessed. The results of incubation of L5178Y cells with eluates of the 5 dialyser

types tested are depicted in Fig. D-8 and Fig. D-9. The relative proliferation is

presented in comparison to the vehicle control, which was normalized to 100% cell

proliferation. The cell proliferation varied between 92% (HF80S, 4 h, ddH2O) and

108% (FX80, 24 h, ddH2O) of the vehicle control. Those variations were statistically

not significant. Therefore it is obvious that neither 4 h eluates (Fig. D-8), nor 24 h

eluates obtained with 17.2% EtOH or ddH2O influenced the cell proliferation in

comparison to the vehicle control. In contrast, incubation with the positive control

MMS (50 µg/ml) reduced the cell proliferation significantly.


83

0

20

40

60

80

100

120

140

Con

trol

FX60

FX60S

FX80

HF80

S

170H

50 µ

g/m

l MM

S

4 h Eluates

Cell

pro

life

rati

on

[%

of

co

ntr

ol]

Fig. D-8 Relative cell proliferation of L5178Y cells after incubation with 4 h eluates of the

dialysers FX60, FX60S, FX80, HF80S and 170H for 24 h. Methyl-methane-

sulfonate (MMS; 50 µg/ml) served as positive control. Results are shown as mean

± S.D. of three independent experiments. *** p ≤ 0.001

0

20

40

60

80

100

120

140

Contro

l

FX60

FX60

S

FX80

HF80S

170H

50 µ

g/ml M

MS

24 h Eluates

Cell

pro

life

rati

on

[%

of

co

ntr

ol]

Fig. D-9 Relative cell proliferation of L5178Y cells after incubation with 24 h eluates of the

dialysers FX60, FX60S, FX80, HF80S and 170H for 24 h. Methyl-methane-

sulfonate (MMS; 50 µg/ml) served as positive control. Results are shown as mean

± S.D. of three independent experiments. *** p ≤ 0.001

3.2 Mitosis Frequency

Second, the influence of eluates on the mitotic frequency of L5178Y cells was

analysed. 24 h incubation with the eluates of varying types of dialysers did not alter

the mitotic frequency of cells compared to the vehicle control - regardless of the

elution conditions. Results of three independent experiments can be seen in Fig.

EtOH eluates

ddH2O eluates

EtOH eluates

ddH2O eluates

***

***


84

D-10 and Fig. D-11. Owing to the varying time spans needed for cell division of

different cell passages, the mitotic frequency is presented as values relative to the

control, not in absolute numbers. The mitotic frequency varied between a relative

mitotic frequency of 0.8 (HF80S, 24 h, 17.2% EtOH) and 1.4 (FX80, 4 h, ddH2O).

These effects were statistically not significant.

0

1

2

3

4

5

Cont

rol

FX60

FX60

S

FX80

HF80S

170H

4 h Eluates

Rel.

Mit

osis

Fig. D-10 Relative mitosis frequency of L5178Y cells after incubation with 4 h eluates of

the dialysers FX60, FX60S, FX80, HF80S and 170H for 24 h. Results are shown

as mean ± S.D. of three independent experiments.

0

1

2

3

4

5

Cont

rol

FX60

FX60

S

FX80

HF80S

170H

24 h Eluates

Rel.

Mit

osis

Fig. D-11 Relative mitosis frequency of L5178Y cells after incubation with 24 h eluates of the

dialysers FX60, FX60S, FX80, HF80S and 170H for 24 h. Results are shown as

mean ± S.D. of three independent experiments.

EtOH

ddH2O

EtOH eluates

ddH2O eluates


85

3.3 Apoptosis

Finally, it was analysed whether eluates are capable of inducing apoptosis. First,

the amount of apoptotic cells after incubation with FX60 and F60S eluates was

determined by annexin V staining and flow cytometry (Fig. D-12a, Fig. D-12b). 24 h

incubation of L5178Y cells with those eluates did not induce apoptosis, while the

positive control (100 µM NaAsO2) was effective. Simultaneously, apoptosis induction

was determined by microscopic analysis. The results obtained by flow cytometry and

microscopy were identical (data not shown). Because the microscopic analysis of

apoptosis could be performed along with analysis for mitosis and MN, the remaining

eluates were analysed by microscopy only. The percentage of apoptotic cells in the

vehicle control was 0.1 ± 0.05%. The percentage of apoptotic cells in the cells treated

with eluate lay between 0 - 0.12% (Fig. D-13, Fig. D-14). Therefore it can be

concluded that eluates did not induce apoptosis in L5178Y cells.

0

5

10

cont

rol

F60SFX60

100

µM A

s

4 h eluates

Ap

op

tos

is [

%]

0

5

10

cont

rol

FX60

F60S

100

µM A

S

24 h eluates

Ap

op

tos

is [

%]

Fig. D-12 Percentage of apoptotic L5178Y cells after incubation with 4 h (a) and 24 h (b) of the

dialysers F60S and FX60 for 24 h. The analysis was performed by flow cytometry.

100 µM. NaAsO2 served as positive control. Results are shown as mean ± S.D. of three

independent experiments.

EtOH

ddH2O

EtOH

ddH2O

a b

D Results Genotoxicity Tests

86

0

1

2

3

4

5

Cont

rol

FX60

FX60

S

FX80

HF80S

170H

4 h Eluates

Ap

op

tosis

[%

]

Fig. D-13 Percentage of apoptotic L5178Y cells after incubation with 4 h eluates of the



0

1

2

3

4

5

Cont

rol

FX60

FX60

S

FX80

HF80S

170H

24 h Eluates

Ap

op

tosis

[%

]

Fig. D-14 Percentage of apoptotic L5178Y cells after incubation with 24 h eluates of the



In summary, none of the eluates exhibited any cytotoxic effect in L5178Y cells.

4 Genotoxicity Tests

After the cytotoxicity tests revealed no evidence for cytotoxicity caused by

eluates the potential genotoxicity of the eluates was analysed by micronucleus test

and comet assay.

EtOH

ddH2O

EtOH

ddH2O


87

4.1 Micronucleus Frequency

As described earlier, incubation with eluates did not influence cell proliferation,

therefore the use of Cyt B in the MN assay was not necessary. None of the 20

eluates tested increased the micronucleus frequency compared to vehicle control

values, while the positive control MMC (0.13 µg/ml) raised the number of

micronucleic cells considerably (Fig. D-15 and Fig. D-16). The relative amount of MN

in the control was normalized to 1. The values of cells treated with eluate varied

between 0.8 (170H, 4 h, ddH2O) and 2.2 (FX80, 4 h, 17.2% EtOH) ± 1. This range

was within the 95% confidence interval of the vehicle control and therefore

statistically not significant.

0

5

10

15

20

25

30

35

Cont

rol

FX60

FX60

S

FX80

HF80S

170H

0.13

µg/m

l MMC

4 h Eluates

Rel.

MN

/1000 c

ell

s

Fig. D-15 Relative number of micronuclei per thousand L5178Y cells after incubation with

4 h eluates of the dialysers FX60, FX60S, FX80, HF80S and 170H for 24 h.

Mitomycin C (MMC; 0.13 µg/ml) served as positive control. Results are shown as

mean ± S.D. of three independent experiments. *** p ≤ 0.001

EtOH

ddH2O

***


88

0

5

10

15

20

25

30

35

Cont

rol

FX60

FX60

S

FX80

HF80S

170H

0.13

µg/m

l MMC

24 h Eluates

Rel.

MN

/1000 c

ell

s

Fig. D-16 Relative number of micronuclei per thousand L5178Y cells after incubation with

24 h eluates of the dialysers FX60, FX60S, FX80, HF80S and 170H for 24 h.

Mitomycin C (MMC; 0.13 µg/ml) served as positive control. Results are shown as

mean ± S.D. of three independent experiments. *** p ≤ 0.001

4.2 Comet Assay

The second genotoxicity test was the comet assay. Incubation of L5178Y cells

with eluates of the dialysers FX60, FX60S, FX80, HF80S or 170H did not increase

the percentage of DNA in tail compared to the ones in the control to a statistically

significant degree (Fig. D-17 and Fig. D-18). The relative amount of DNA in tail varied

between 0.8 (HF80S, 4 h, 17.2% EtOH) and 1.9 (FX60S, 24 h, ddH2O) compared to

1 in the vehicle control. The was a slight tendency for increased genomic damage

after incubation with 17.2% EtOH eluates of FX60 and F60S dialysers; however,

statistical significance was not reached. As expected the positive control MMS

(50 µg/ml) increased the amount of DNA in tail considerably.

EtOH

ddH2O

***


89

0

2

4

6

8

10

12

Contro

l

FX60

FX60S

FX80

HF80S

170H

50 µ

g/ml M

MS

4 h Eluates

Rel.

am

ou

nt

of

DN

A i

n t

ail

Fig. D-17 Relative DNA damage of L5178Y cells detected by the comet assay after

incubation with 4 h eluates of the dialysers FX60, FX60S, FX80, HF80S and 170H

for 24 h. Methyl-methane-sulfonate (MMS; 50 µg/ml) served as positive control.

Results are shown as mean ± S.D. of three independent experiments. *** p ≤ 0.001

0

2

4

6

8

10

12

Contro

l

FX60

FX60

S

FX80

HF80S

170H

50 µ

g/ml M

MS

24 h Eluates

Rel.

am

ou

nt

of

DN

A i

n t

ail

Fig. D-18 Relative DNA damage of L5178Y cells detected by the comet assay after

incubation with 24 h eluates of the dialysers FX60, FX60S, FX80, HF80S and 170H

for 24 h. Methyl-methane-sulfonate (MMS; 50 µg/ml) served as positive control.

Results are shown as mean ± S.D. of three independent experiments. *** p ≤ 0.001

4.3 Test for Estrogenic Activity

Because BPA possesses estrogenic activity and it can be detected in dialyser

eluates, tests for estrogenic activity (E-screens) were performed. The E-screens were

conducted with MCF-7 cells which are more sensitive towards solvents in media than

L5178Y cells. Therefore only 50 µl of 17.2% EtOH eluates or 100 µl of ddH2O eluates

EtOH

ddH2O

EtOH

ddH2O

***

***


90

could be added. This led to a 50% decrease of eluate concentration in the media

compared to the toxicity testing.

4.3.1 E-Screen

Incubation of MCF-7 cells with 17.2% EtOH eluates of the dialysers FX60,

FX60S, FX80, HF80S and 170H (24 h eluates only) increased the proliferation by

30% - 60%. Incubation with ddH2O eluates of the dialysers FX60, FX60S, FX80,

HF80S and 170H (24 h eluates) increased the proliferation slightly, but not in a

statistically significant way (Fig. D-19).

0

100

200

300

400

500

Cont

rol

FX60

FX60

S

FX80

HF80S

170H

1 µM

BPA

24 h Eluates

Cell

pro

life

rati

on

[%

co

ntr

ol]

Fig. D-19 Relative proliferation of MCF-7 cells after 8 days incubation with 24 h eluates of the

dialysers FX60, FX60S, FX80, HF80S and 170H. Bisphenol A (1µM) served as

positive control. Results are shown as mean ± S.D. of three independent

experiments. * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001

In order to decide whether the increase of proliferation was due to BPA in the

eluates, a calibration curve of pure BPA in the range of 0.1 - 1000 nM was

established (Fig. D-20). The proliferation of MCF-7 cells started to increase

significantly after incubation with 31.6 nM BPA. This equals 7.2 ng/ml BPA. This

increase of proliferation was dose-dependent.

*

**

EtOH

ddH2O

* * * *

D Results Summary of the Toxicity Testing

91

0

100

200

300

400

500

0,1 1 10 100 1000

[c] BPA nM

Ce

ll p

roli

fera

tio

n [

% c

on

tro

l]

Fig. D-20 Relative proliferation of MCF-7 cells after incubation with bisphenol A for 8 days.

Results are shown as mean ± S.D. of three independent experiments.

5 Summary of the Toxicity Testing

To summarize: in the range of concentration tested eluates did not cause any

effect in the cytotoxicity and genotoxicity test of this study (Tab. D-5). The only effect

was an increase of proliferation of estrogen sensitive MCF-7 cells by 17.2% ethanol

eluates.

D Results Uremic Toxins

92

Test system Eluent Time FX60 F60S FX80 HF80S 170H

4 h - - - - - ddH2O

24 h - - - - -

4 h - - - - - Proliferation

EtOH 24 h - - - - -

4 h - - - - - ddH2O

24 h - - - - -

4 h - - - - - Mitosis frequency

EtOH 24 h - - - - -

4 h - - - - - ddH2O

24 h - - - - -

4 h - - - - - Apoptosis

EtOH 24 h - - - - -

4 h - - - - - ddH2O

24 h - - - - -

4 h - - - - -

Micronucleus frequency

EtOH 24 h - - - - -

4 h - - - - - ddH2O

24 h - - - - -

4 h - - - - - Comet-Assay

EtOH 24 h - - - - -

ddH2O 24 h - - - - - E-Screen

EtOH 24 h + + + + +

Tab. D-5 Summary of the toxicity testing.

6 Uremic Toxins

Several uremic toxins with suspected genotoxicity have been analysed in this

study: Hcy and its derivate Hcy-T as well as leptin and AGEs.

6.1 Homocysteine and Homocysteine-Thiolactone

6.1.1 Cytotoxicity Testing

The uremic toxin studied most intensively was Hcy. Two cell lines were chosen

for the analysis: L5178Y cells and HL60 cells. L5178Y cells were selected because

they are an established cell line for genotoxicity testing. HL60 cells were used

because they are known to be sensitive towards oxidative stress, an expected effect


93

of Hcy. Toxicity tests of the Hcy derivate Hcy-T were conducted with L5178Y cells

only.

6.1.1.1 Proliferation

In order to determine the concentration at which Hcy displays cytotoxicity in

L5178Y cells range-finding experiments were performed. The Hcy concentrations

varied from 0.1 mM to 10 mM. Two incubation times were chosen: 24 h and 120 h,

the latter should resemble long term exposure which is relevant for dialysis patients.

Starting at 3.16 mM Hcy 120 h incubation caused a considerable reduction of

proliferation (Fig. D-21). The same effects could be observed after 24 h of exposure,

but only at concentrations higher than 3.16 mM.

0

20

40

60

80

100

120

Cont

rol

0.1

mM H

cy

0.31

6 mM

Hcy

1 m

M H

cy

3.16

mM

Hcy

10 m

M H

cy

MM

CCell

pro

life

rati

on

[%

co

ntr

ol]

24 h

120 h

Fig. D-21 Proliferation of L5178Y cells after incubation with homocysteine for 24 h (lighter

bars) or 120 h (black bars), respectively. Mitomycin C (MMC; 125 ng/ml) served

as positive control. (Single experiment)

Subsequently, improved cytotoxicity tests were conducted with L5178Y and HL60

cells. A 24 h incubation with 4 mM Hcy decreased cell proliferation of L5178Y cells to

a statistically significant degree. Based on their longer cell cycle HL60 cells were

incubated for 48 h. The proliferation of HL60 cells decreased slightly but not

significantly up to concentrations of 5 mM (Fig. D-22).


94

0

20

40

60

80

100

120

140

Cont

rol

1 m

M H

cy

2 m

M H

cy

3 m

M H

cy

4 m

M H

cy

5 m

M H

cy

MM

CCell

pro

life

rati

on

[%

of

co

ntr

ol]

L5178Y

HL60

**

****

Fig. D-22 Proliferation of L5178Y cells (dark bars) and HL60 cells (bright bars) relative to the

vehicle control after incubation with homocysteine for 24 h (L5178Y) or 48 h (HL60)

respectively. Mitomycin C (MMC; 125 ng/ml) served as positive control. Results are

shown as mean ± S.D. of three independent experiments * p ≤ 0.05, ** p ≤ 0.01,

*** p ≤ 0.001

Hcy-T was more cytotoxic than Hcy. A statistical significant reduction of

proliferation could already be observed at 1 mM Hcy-T already (Fig. D-23).

0

20

40

60

80

100

120

Contro

l

1 m

M H

cy-T

2 m

M H

cy-T

3 m

M H

cy-T

4 m

M H

cy-T

5 m

M H

cy-T

MM

C

Cell

pro

life

rati

on

[%

of

co

ntr

ol]

*

**

**

**

*

***

Fig. D-23 Proliferation of L5178Y cells relative to the vehicle control after incubation with

homocysteine-thiolactone (Hcy-T) for 24 h. Mitomycin C (MMC; 125 ng/ml) served

as positive control. Results are shown as mean ± S.D. of three independent

experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001


95

6.1.1.2 Mitosis

In parallel to cell proliferation the number of mitotic cells was analysed

microscopically. The mitotic frequency of L5178Y cells decreased statistically

significant way at 5 mM Hcy, a concentration which was also cytotoxic. In HL60 cells

a tendency towards a reduced percentage of mitotic cells could be observed at 5 mM

Hcy (Fig. D-24). However this tendency was statistically non-significant.

0

5

10

15

20

Cont

rol

1 m

M H

cy

2 m

M H

cy

3 m

M H

cy

4 m

M H

cy

5 m

M H

cy

MM

C

Mit

oti

c c

ell

s [

%]

L5178Y

HL60

****

Fig. D-24 Percentage of cells undergoing mitosis in L5178Y cells (dark bars) and HL60 cells

(bright bars) after incubation with homocysteine for 24 h (L5178Y) or 48 h (HL60)

respectively. Mitomycin C (MMC; 125 ng/ml) served as positive control. Results are

shown as mean ± S.D. of three independent experiments * p ≤ 0.05, ** p ≤ 0.01,

*** p ≤ 0.001

6.1.1.3 Apoptosis

In addition, the percentage of apoptotic cells was determined microscopically.

Hcy did not induce apoptosis in L5178Y cells up to concentrations of 5 mM, whereas

it induced apoptosis in HL60 cells (Fig. D-25). This effect was statistically significant

at concentrations of 4 mM Hcy and higher. At 5 mM the number of apoptotic cells

was roughly tripled compared to the vehicle control.


96

-10

0

10

20

30

40

50

Cont

rol

1 m

M H

cy

2 m

M H

cy

3 m

M H

cy

4 m

M H

cy

5 m

M H

cy

MM

C

Ap

op

tosis

[%

]L5178Y

HL60

***

***

Fig. D-25 Percentage of cells undergoing apoptosis in L5178Y cells (dark bars) and HL60

cells (light bars) after incubation with homocysteine for 24 h (L5178Y) or 48 h

(HL60) respectively. Mitomycin C (MMC; 125 ng/ml) served as positive control.

Results are shown as mean ± S.D. of three independent experiments * p ≤ 0.05,

** p ≤ 0.01, *** p ≤ 0.001

The possible pro-apoptotic effects of Hcy-T were only analysed in L5178Y cells.

A stitistically significant and dose-dependent induction of apoptosis was observed at

3 mM - 5mM Hcy-T (Fig. D-26).

0

5

10

15

20

Cont

rol

1 m

M H

cy-T

2 m

M H

cy-T

3 m

M H

cy-T

4 m

M H

cy-T

5 m

M H

cy-T

Ap

op

tosis

[%

]

*

**

**

Fig. D-26 Percentage of cells undergoing apoptosis in L5178Y cells after incubation with

homocysteine-thiolactone (Hcy-T) for 24 h respectively. Mitomycin C (MMC;

125 ng/ml) served as positive control. Results are shown as mean ± S.D. of three

independent experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001


97

6.1.2 Genotoxicity Tests

6.1.2.1 Micronucleus Assay

Simultaneously to the range-finding experiments for cytotoxicity the range-finding

experiments for genotoxicity were conducted. The micronucleus frequency started to

increase after incubation with 3.16 mM Hcy for 24 h (Fig. D-27). Incubation for 5 days

did increase the number of micronuclei even further but did not lower the threshold.

0

20

40

60

80

100

Cont

rol

0.1

mM H

cy

0.31

6 mM

Hcy

1 m

M H

cy

3.16

mM

Hcy

10 m

M H

cy

MM

C

MN

/ 1

000 c

ell

s

24 h

120 h

Fig. D-27 Micronucleus induction in L5178Y cells after incubation with homocysteine for 24 h

(brighter bars) or 120 h (black bars) respectively. Mitomycin C (MMC; 125 ng/ml)

served as positive control.

Subsequently, improved micronucleus assays showed a significant increase of

MN after 24 h incubation of L5178Y with 3 mM Hcy. This effect was even more

pronounced at higher concentrations (Fig. D-28).

In HL60 cells a significant increase of the micronucleus frequency could only be

observed at 3 mM Hcy, not at higher or lower concentrations. As there was no dose-

dependency a coincidental finding cannot be ruled out.


98

0

20

40

60

80

100

Cont

rol

1 m

M H

cy

2 m

M H

cy

3 m

M H

cy

4 m

M H

cy

5 m

M H

cy

MM

C

MN

/ 1

000 c

ell

sL5178Y

HL60

***

**** **

***

Fig. D-28 Micronucleus induction in L5178Y cells (dark bars) and HL60 cells (bright bars)

after incubation with homocysteine for 24 h (L5178Y) or 48 h (HL60) respectively.

Mitomycin C (MMC; 125 ng/ml) served as positive control. Results are shown as

mean ± S.D. of three independent experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Apart from the induction of MN, Hcy also reduced the mitotic rate of L5178Y

cells, therefore additional MN assays with Cyt B were performed. However, the

results did not differ from the MN tests described above.

The Hcy derivate Hcy-T elevated the MN frequency at 1 mM, the same

concentration which reduced cell proliferation. The number of MN increased in a

dose-dependent manner (Fig. D-29).

Fig. D-29 Micronucleus induction in L5178Y cells after incubation with homocysteine-

thiolactone (Hcy-T) for 24 h. Mitomycin C (MMC; 125 ng/ml) served as positive

control

0

20

40

60

80

100

Cont

rol

1 m

M H

cy-T

2 m

M H

cy-T

3 m

M H

cy-T

4 m

M H

cy-T

5 m

M H

cy-T

MM

C

MN

/ 1

000 c

ell

s

**

**

*

*

***

**

*


99

To evaluate whether MN induction is cell-type specific for L5178Y cells, MN tests

with additional cell lines (CaCo, TK6 and LLC-PK1) were performed (Fig. D-30). Hcy

and Hcy-T increased the MN frequency at 4 mM, the same range at which it induced

MN in L5178Y. Therefore the genotoxicity seems to occur in a broad range of cell

lines.

0

20

40

60

80

100

Contro

l

4 m

M H

cy

4 m

M H

cy-T

MN

/ 1

000 c

ells

Caco

TK6

LLC-PK1

Fig. D-30 Micronucleus induction in CaCo cells (black bars) TK6 cells (grey bars) and LLC-

PK1 cells after incubation with homocysteine and homocysteine-thiolactone

(Hcy-T) for 48 h.

6.1.2.2 Comet Assay

While the MN frequency test yielded evidence for genotoxicity of Hcy and Hcy-T,

the second genotoxicity test, the comet assay, did not support this finding. Hcy did

not increase the relative amount of DNA in tail of L5178Y or HL60 cells up to

cytotoxic concentrations (Fig. D-31); the same was true for Hcy-T (Fig. D-32).


100

0

5

10

15

20

Cont

rol

1 m

M H

cy

2 m

M H

cy

3 m

M H

cy

4 m

M H

cy

5 m

M H

cy

MM

S

Rela

tive a

mo

un

t o

f D

NA

in

tail

*L5178Y

HL60 *

Fig. D-31 DNA damage analysed by comet assay (relative percentage of DNA in tail) after

incubation of L5178Y cells (dark bars) and HL60 cells (bright bars) with

homocysteine for 24 h. Methyl-methane-sulfonate (MMS; 50 µM) served as


experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

0

5

10

15

20

Cont

rol

1 m

M H

cy-T

2 m

M H

cy-T

3 m

M H

cy-T

4 m

M H

cy-T

5 m

M H

cy-T

MM

SRela

tive a

mo

un

t o

f D

NA

in

tail

*


incubation of L5178Y cells with homocysteine-thiolactone (Hcy-T) for 24 h. Methyl-

methane-sulfonate (MMS; 50 µM) served as positive control. Results are shown as

mean ± S.D. of three independent experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

DNA damage, like DNA strand breaks, is normally detectable by comet assay.

However, DNA strand breaks can be repaired by enzymes and may therefore be

overlooked in a 24 h assay. In order to rule out this possibility, short-term comet

assays (incubation period 2-24 h) were conducted on L5178Y cells. No increased


101

DNA damage could be observed after incubation with Hcy (Fig. D-33) or Hcy-T (Fig.

D-34).

0

5

10

15

20

2 h

4 h

8 h

24 h

MM

S

Rela

tive a

mo

un

t o

f D

NA

in

tail

Control

5 mM Hcy


incubation of L5178Y cells with 5 mM homocysteine for 2 - 24 h. Methyl-methane-

sulfonate (MMS; 50 µM) served as positive control (single experiment).

0

5

10

15

20

2 h

4 h

8 h

24 h

MM

S

Rela

tive a

mo

un

t o

f D

NA

in

tail

Control

5 mM Hcy-T


incubation of L5178Y cells with 5 mM homocysteine-thiolactone (Hcy-T) for

2 - 24 h. Methyl-methane-sulfonate (MMS; 50 µM) served as positive control

(single experiment).


102

6.1.3 Oxidative Stress

6.1.3.1 Oxidative Stress & Micronuclei

One possible reason for increased genomic damage is oxidative stress. In order

to analyse whether Hcy induces MN by causing oxidative stress, the radical

scavenger N-acetylcysteine (NAC) was added to cells challenged by Hcy. Addition of

NAC did not reduce the MN frequency of L5178Y cells induced by Hcy or Hcy-T

(Fig. D-35).

0

20

40

60

80

100

Contro

l

NAC

3 m

M

3 m

M +

NAC

MM

C

MN

/ 1

000 c

ell

s

Hcy

Hcy-T

Fig. D-35 Micronucleus frequency found in L5178Y cells after incubation with 3 mM

homocysteine or homocysteine-thiolactone (Hcy-T) and N-acetylcysteine for 24 h.

(single experiment)

6.1.3.2 Flow Cytometric Analysis of Oxidative Stress

In order to determine whether Hcy induces oxidative stress at all, flow cytometric

analyses of HL60 cells after incubation with 3 mM Hcy were conducted. 0.5 mM H2O2

served as positive control. To analyse whether Hcy can modulate oxidative stress,

experiments of co-incubation with H2O2 and Hcy were performed. Oxidative stress

can be very short-lived, therefore incubation periods from 30 min to 24 h were

analysed. During this period Hcy did not induce oxidative stress observable by DCF

fluorescence. On the contrary: Hcy was able to reduce oxidative stress induced by

H2O2 (Fig. D-36, Fig. D-38).


103

0

20

40

60

80

100

120

0 m

in

30 m

in

60 m

in

90 m

in

120 m

in

H2O

2

Incubation period

Rela

tive D

CF

flu

ore

scen

ce

3 mM Hcy + 0.5 mM H2O2

0.5 mM H2O2

3 mM Hcy

Fig. D-36 Oxidative stress level measured by relative DCF fluorescence after 30 to 120 min

incubation of HL60 cells with 3 mM homocysteine (black bars) or pre-treated with

homocysteine and challenged with H2O2 (grey bars). Results are shown as mean

± S.D. of three independent experiments.

0

20

40

60

80

100

120

0 h

4 h

24 h

H2O

2

Incubation period

Rela

tive D

CF

flu

ore

scen

ce

3 mM Hcy + 0.5 mM H2O2

0.5 mM H2O2

3 mM Hcy

Fig. D-37 Oxidative stress level measured by relative DCF fluorescence after 4 to 24 h

incubation of HL60 cells with 3 mM homocysteine (black bars) or pre-treated with

homocysteine and challenged with H2O2 (grey bars). Results are shown as mean

± S.D. of three independent experiments.

6.1.4 GSH

One possible explanation for the anti-oxidative effect of Hcy is the conversion of

Hcy to the cellular antioxidant GSH. Therefore the GSH levels of HL60 and L5178Y

cells were analysed by GSH/GSSG assay.


104

After exposure to 3 mM Hcy, the GSH content of L5178Y and HL60 cells

increased within 30 min (Fig. D-38, Fig. D-39). The GSH content of L5178Y cells

doubled within 18 h and decreased later on. The GSH content of HL60 cells reached

its peak already after 2 h and dropped back to control values within the next 22 h.

0

50

100

150

200

250

300

350

Cont

rol

0.5

h1

h2

h4

h18

h24

h

Incubation Period

Rela

tive G

SH

level

[%]

*

*

**

*

Fig. D-38 Relative amount of GSH in L5178Y cells after incubation with 3 mM homocysteine

for 0.5 to 24 h. Results are shown as mean ± S.D. of three independent

experiments * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001

0

50

100

150

Cont

rol

0.5

h1

h2

h4

h18

h

24 h

Incubation period

Rela

tive G

SH

level

*

***

*

Fig. D-39 Relative amount of GSH in HL60 cells after incubation with 3 mM homocysteine for

0.5 to 24 h. Results are shown as mean ± S.D. of three independent experiments

* p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001


105

6.1.5 Methylation

Another potential mechanism for MN induction is the change of overall DNA

methylation. Therefore DNA methylation of L5178Y cells was analysed by flow

cytometry and HPLC-MS/MS after incubation with Hcy. No significant changes could

be detected by either method, while the positive control 5-azacytidine reduced overall

DNA methylation dramatically (Fig. D-40). Additionally, the influence of Hcy-T on

overall DNA methylation was determined by HPLC-MS/MS. No significant effect

could be observed (Fig. D-41).

Flow cytometry

0

20

40

60

80

100

120

140

160

Cont

rol

3 m

M H

cy

5 m

M H

cy

250 nM

5-A

za-C

1250

nM

5-A

za-C

Rela

tive D

NA

meth

yla

tio

n

*

***

Fig. D-40 Flow cytometric analysis of DNA-cytosine methylation in L5178Y cells after 72 h

exposition of homocysteine. The results are shown as relative DNA-cytosine-

methylation compared to untreated cells. 5-Azacytidine (5- Aza-C) served as


experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001


106

0

20

40

60

80

100

120

140

160

Cont

rol

3 m

M H

cy

5 m

M H

cy

250 nM

5 A

za-d

C

Rela

tive D

NA

-cyto

sin

e-m

eth

yla

tio

n

**

Hcy

Hcy-T

Fig. D-41 Analysis of DNA-cytosine methylation in L5178Y cells after 72 h exposition of

homocysteine by HPLC-MS/MS. The results are shown as relative DNA-cytosine-

methylation compared to untreated cells. 5-Azacytidine (5-Aza-C) served as


experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

6.1.6 BrdU

Microscopic analysis of Hcy-treated cells revealed a reduced percentage of

mitotic cells. The kind of mitotic interference was specified by BrdU incorporation

assay. A 12 h incubation of L5178Y cells with up to 5 mM Hcy did not alter the

percentage of cells in specific phases significantly (Fig. D-42). However, after 24 h

incubation with 5 mM Hcy the percentage of cells in the G1-Phase was reduced

significantly, while the amount of cells in the S-Phase increased (Fig. D-43).

Incubation with 3 mM Hcy had the same effect, but did not reach significance.


107

0%

20%

40%

60%

80%

100%

0 m

M H

cy

3 m

M H

cy

5 mM

Hcy

Pe

rce

nta

ge

of

cells in

sp

ecif

ic

cell-c

yc

le p

has

eS-Phase

G1-Phase

G2/M-Phase

Sub-G1 peak

Fig. D-42 Cell cycle analysis of L5178Y cells by BrdU incorporation assay after 12 h

exposure to homocysteine. The results are given as percentage of cells in the

S-Phase, G1-phase, G2-M phase and sub-G1 peak. Results are shown as mean

± S.D. of three independent experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

0%

20%

40%

60%

80%

100%

0 m

M H

cy

3 m

M H

cy

5 m

M H

cy

Pe

rce

nta

ge

of

ce

lls

in

sp

ec

ific

ce

ll-c

yc

le p

ha

se

S-Phase

G1-Phase

G2/M-Phase

Sub-G1 peak

*

Fig. D-43 Cell cycle analysis of L5178Y cells by BrdU incorporation assay after 24 h

exposure to homocysteine. The results are given as percentage of cells in the

S-Phase, G1-phase, G2-M phase and sub-G1 peak. Results are shown as mean

± S.D. of three independent experiments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001


108

6.2 Leptin

6.2.1 Cytotoxicity Testing

L5178Y cells were incubated with 0.1 to 10 µg/ml leptin. Those concentrations

had no effect on the cell proliferation, mitotic frequency (data not shown) or apoptosis

analysed by flow cytometry (Fig. D-44)

0

1

2

3

4

5

Cont

rol

0.1

µg/m

l Lep

tin

0.31

6 µg/

ml L

eptin

1 µg

/ml L

eptin

3.16

µg/m

l Lep

tin

10 µ

g/ml L

eptin

100 µM

As

Ap

op

tosis

[%

]

Fig. D-44 Percentage of apoptotic L5178Y cells after 24 h incubation 0.1 – 10 µg/ml NaAsO2.

6.2.2 Genotoxicity Testing

The genotoxic effect of leptin was analysed by MN frequency test (Fig. D-45) and

by comet assay (Fig. D-46). Leptin did not influence the MN frequency up to

concentration of 10 µg /ml. However, leptin induced DNA damage by comet assay at

concentrations as low as 1 µg/ml. As it was not analysed which kind of Ob receptors

are presented on the cell surface of L5178Y cells, it could not be elucidated whether

the DNA damage was mediated by receptor, or whether leptin was ingested by the

cell and interacted directly with DNA.


109

0

20

40

60

80

100

120

Cont

rol

0.1

µg/m

l Lep

tin

0.31

6 µg/

ml L

eptin

1 µg

/ml L

eptin

3.16

µg/m

l Lep

tin

10 µ

g/ml L

eptin

MM

C

MN

/ 1

000 c

ell

s

Fig. D-45 Micronucleus induction in L5178Y cells after incubation with leptin for 24 h.

Mitomycin C (MMC; 125 ng/ml) served as positive control. Results are shown as

mean values between two independent experiments.

0

10

20

30

Cont

rol

0.1

µg/m

l Lep

tin

0.31

6 µg/

ml L

eptin

1 µg

/ml L

eptin

3.16

µg/m

l Lep

tin

10 µ

g/ml L

eptin

MM

S

Rela

tive a

mo

un

t o

f D

NA

in

tail


incubation of L5178Y cells with leptin for 24 h. Methyl-methane-sulfonate (MMS;

50 µM) served as positive control. Results are shown as mean of three

independent experiments ± standard deviation * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

As the effect of leptin could only be detected in the comet assay and what is

more, as this effect was observed only at concentrations 1000 times higher than the

ones in patients, further experiments were abandoned.

* *

*

*


110

6.3 Advanced Glycation End Products

Finally, the genotoxic capacity of AGEs was examined. This was mainly done on

porcine proximal tubule cells LLC-PK1. Incubation with freshly synthesized AGEs

(CML and MGO) did not influence cell proliferation or number of apoptotic cells, but it

induced genomic damage detectable by comet-assay (Fig. D-47). However, the

AGEs synthesized by ourselves lost their activity already after one or two weeks,

even if stored at -80°C. There were also huge differences between AGEs produced

from different BSA batches. These problems prevented the averaging of three

consecutive experiments.

0

10

20

30

40

50

BSA

-Con

trol

1 m

g/ml C

ML

1 m

g/ml M

GO

50 µ

M M

MS

Rela

tive a

mo

un

t o

f D

NA

in

tail

Fig. D-47 Exemplary comet assay of LLC-PK1 cells incubated with AGEs for

24 h.

6.4 Summary of Toxic Effects of Uremic Toxins

A summary of toxic effects of uremic toxins analysed in this study is given in Tab.

D-6. Additional information on three further uremic toxins - indole-3-acetic acid,

indoxyl sulfate and methylguanidine - is presented in this table. This information was

gathered during the master thesis of co-worker Birgit Werner (Werner 2005).

D Results Effects of Patient Serum

111

Hcy Leptin AGEs Indole-3-

acetic acid Indoxyl sulfate

Methylguanidine

Cytotoxicity + - - + + +

Mitotic frequency

+ - n.d. n.d. n.d. n.d.

Apoptosis - - - n.d. n.d. n.d.

Micronuclei + - n.d. - (+) +

Comet-Assay - + + + - -

Tab. D-6 Summary of cytotoxic/genotoxic effects of uremic toxins on L5178Y or LLC-PK1

cells; +: effect; -: no effect; n.d.: not determined.

7 Effects of Patient Serum

Several uremic toxins exhibited genotoxic features but none of them at

concentrations or to an extent which could by itself explain the increased genomic

damage in HD patients. Therefore, we analysed whether the increased genomic

damage could directly be related to the sum of the known and unknown substances

present in the patient serum. Serum samples of HD patients who displayed an

increased MN frequency (as analysed by another study (Treutlein, to be submitted),

were prepared. Additionally a 10 kDa filtrate of the serum was obtained. This filtrate

corresponds to the filtrate of average dialysers. Thereafter, L5178Y cells were

incubated with serum or 10 kDa filtrate for 24 h.

In order to mimic the real life situation the patient serum in the media should be

as highly concentrated as possible. To evaluate the maximum amount of serum

which could be added to L5178Y cells in general without influencing them,

preliminary experiments with horse serum, were performed. Addition of up to 30%

horse serum to the cell culture media did not alter cell proliferation or appearance.

Unfortunately, L5178Y cells tolerated human serum not in the same way. The

addition of 20% patient serum to the normal cell media (containing 10% horse serum)

killed all of the L5178Y cells. Heat inactivation of the complement system did not

reduce the cytotoxicity.

L5178Y cell resisted to the addition of up to 2% patient serum without showing

severe cytotoxic effect (except for the serum of patient one). However, with 3 out of 4

serum samples there was also no increase of MN frequency, even if incubation was

maintained for up to one week (Fig. D-48 and Fig. D-49). However, whether this was

D Results Effects of Patient Serum

112

due to the strong dilution or the lack of genotoxic capacity could not be elucidated.

Solely the plasma of patient one doubled the MN frequency, but it also caused

cytotoxicity, the genotoxic effect may be secondary.

Additionally, cells were incubated with 10kDa filtrate. Concentration up to 20%

filtrate hardly influenced the cell proliferation or MN frequency (Fig. D-48 and Fig.

D-49).

0

20

40

60

80

100

120

140

Cont

rol

Hors

e ser

um

Pat

ient

1

Pat

ient

2

Pat

ient

3

Pat

ient

4

Cell

pro

life

rati

on

[%

of

co

ntr

ol]

10 kDa filtrate

Patient serum

Fig. D-48 Proliferation after incubation of L5178Y cells after incubation of L5178Y cells with

2% patient serum for 1 week (black bars) or with 20% 10 kDa filtrate of the

patient serum for 24 h (grey bars).

Fig. D-49 Micronucleus induction in L5178Y cells after incubation of L5178Y cells with 2%

patient serum for 1 week (black bars) or with 20% 10 kDa filtrate of the patient

serum for 24 h (grey bars).

E Discussion Dialysers

113

E Discussion

Dialysis patients suffer from increased cancer incidence and increased genomic

damage (Maisonneuve, Agodoa et al. 1999; Stopper, Meysen et al. 1999; Stopper,

Boullay et al. 2001; Teschner, Garte et al. 2002; Stewart, Buccianti et al. 2003;

Vajdic, McDonald et al. 2006). Many causes have been discussed in the literature.

However, this study focused on only two:

1. The potentially genotoxic effect of substances leaching from dialysers, and

2. the possible genotoxic effect of selected uremic toxins

1 Dialysers

Optical evaluation of freeze-dried eluates proved clearly that substances are

leaching from blood circuits containing dialysers and tubing. HPLC analysis of

eluates confirmed this observation. A comparison between the full ion spectra of

eluates and pure eluent (ddH2O or 17.2% EtOH) showed significant changes which

could not be attributed to one single substance. Therefore we concluded that several

different substances leach from dialysers. It was most likely that these substances

consist of mechanical abrasion of the tubing by the flexible-tube pump, production

residue like pore filler material glycerol as well as BPA from dialysers or platicizers

like DEHP from the tubing.

This is in line with previous studies which report DEHP leaching from PVC blood-

tubing (Flaminio, Bergia et al. 1988; Faouzi, Dine et al. 1999; Dine, Luyckx et al.

2000) or BPA leaching from synthetic dialyser membranes (Haishima, Hayashi et al.

2001; Yamasaki, Nagake et al. 2001; Murakami, Ohashi et al. 2007).

1.1 BPA

BPA was detected in all eluates. The extrapolated amounts of leaching BPA

ranged from 6.4 ng/ dialyser to 4.3 µg/ dialyser. Several factors contributed to this

broad range of leaching BPA: (1.) the type of dialyser, (2.) the batch, (3.) the choice

of eluent and (4.) the time period for which the extraction was performed.

As expected a prolongation of the extraction time resulted in an increase of BPA

leaching from the blood circuit. This increase was between 10 - 50% of the overall

amount except for 2 cases (FX80, Lot-No NCX01130, ddH2O and 170H, Lot-No S-

4402-H-01, 17.2% EtOH) in which the amount stayed nearly the same.


114

The choice of eluent had a much greater impact on the amount of leaching BPA.

The BPA concentration of 17.2% EtOH eluates was up to 47 times higher than the

one in comparable ddH2O eluates. This was a reasonable finding because the

extraction capacity of a water/ethanol mixture is much higher in regard to BPA.

The third factor that influenced the amount of leaching BPA - the type of dialyser

- was also anticipated. In case of dialysers which consisted of the same material for

housing and membranes but differed in the surface area (FX60 vs FX80 and F60S vs

HF80S), the amount of extracted BPA was higher in the dialysers with the larger

surface area.

More surprising were the huge differences of leaching BPA between single

batches of FX80 and FX60 dialysers. Errors of eluate handling, e.g. by insufficiently

cleaned glassware, can be ruled out because eluates of the same batch were

produced on different days using new equipment. It can therefore be assumed that

quality differences during the manufacturing process and differences in the

originating polysulfone-granules before extrusion are the underlying cause. In

addition the variations of estimated BPA content leaching from dialysers of those

batches was more than 40%, while it was generally below 20% in batches from which

low amount of BPA were leaching.

Given 17.2% EtOH has similar extraction properties towards BPA as bovine

serum - and hence to human serum – those eluates have been used to estimate

human exposure. Assuming the worst case an absolute amount of 4.3 µg BPA would

be leaching into the blood of the dialysis patient per dialysis session (FX80 dialyser,

Lot No: NCX01130, 24 h elution, 17.2% EtOH). If one bases the estimation of

additional body burden on an average adult person (5 l blood volume, 70 kg body

weight) this leads to an additional 0.86 ng BPA/ml blood or 61.4 ng BPA/kg body

weight respectively. Presumably, this calculation overestimates the amount of BPA

because a dialysis session lasts only 4 h, not 24 h. This reduction leads to a 21%

decrease of leaching BPA in this type of dialyser (Table D-3, page 81).

Some previous studies already evaluated the amount of BPA released from

hemodialysers. Haishima et al. analysed four different dialyser types composed of a

polycarbonate or polystyrene housing in combination with cellulose acetate or

polysulfone hollow fibres (Haishima, Hayashi et al. 2001). They detected 3.78 to

141.8 ng BPA/module when using ddH2O as a solvent, 140.1 to 2,090 ng

BPA/module when using bovine serum as solvent or 153.3 to 2,090 ng BPA/module


115

when using 17.2 EtOH as solvent. The values obtained with ddH2O correspond to our

findings (6.4 to 71.3 ng BPA/ module). The amounts of BPA extracted by 17.2%

ethanol are also within the range reported by Haishima et al. for all but a single batch

of FX80 dialysers (Lot No: NCX01130). This batch released roughly twice as much

BPA than the highest report by Haishima. As Haishima et al. performed the extraction

at room temperature and not - like we did - at 37°C - 38°C (the temperature normally

used during dialysis), washed the hemodialysers 3 times prior to BPA extraction,

used a slower flow rate (19 ml/min instead of 230 ml/min) and a shorter extraction

period (16 h instead of 24 h), this slight discrepancy is easily explained.

Yet another group detected a maximum of 1.14 ng BPA/ dialyser or nothing in the

effluent of 5 different dialysers (Yamasaki, Nagake et al. 2001). However, they

neither circulated the water, nor did they specify how long it stayed inside the dialyser

or which temperature was used. They also failed to detect BPA in whole blood

samples of one HD group. This is in contrast to the various studies which report BPA

even in blood samples of the normal population (see below). Furthermore no

information on the HPLC method used is given; therefore it is not possible to

compare it to our results.

A recent study reports rather high concentrations of BPA (83.3 ng/10 mg hollow

fibres) released by polysulfone or PEPA polyester-polymeralloy (Nikkiso, Japan)

(122.5 ng/10 mg hollow fibre) hollow fibres (Murakami, Ohashi et al. 2007). However,

the authors obtained these results by crushing the hollow fibres and dissolving them

in DMSO. Therefore these results are not comparable to the in vivo situation or our

data.

The same study also analysed the amount of BPA leaching from dialysers with

polysulfone membranes directly into the blood of 15 HD patients. The BPA level of

those patients increased from 4.83 ± 1.94 ng/ml blood prior to dialysis to 6.62 ±

3.09 ng/ml thereafter (Murakami, Ohashi et al. 2007). This equates to an additional

BPA body burden of 1.79 ng/ml blood which is roughly twice as much as our worst

case estimation. However, another experiment with the same patients and the same

dialysers resulted in an increase of only 0.18 ng BPA/ml blood during dialysis. This

corresponds to 20 % of the increase estimated by our worst case scenario.

Unfortunately, the paper does not explain the discrepancy between their

measurements. It can therefore be assumed that the amount of BPA released was


116

midway between 0.18 and 1.8 ng/ml blood, which is also what our experiments

predicted.

In order to evaluate the risk which BPA poses to HD patients, the amount has to

be compared to the BPA burden of average humans. Over the last few years more

than a dozen studies have measured BPA in blood of men and women from several

countries at different ages. The analysis included a variety of analytical methods like

ELISA, LC-MS, GC-MS or HPLC. Depending on the method, the BPA content ranged

from 0.3 – 20 ng/ml blood (Inoue, Kato et al. 2000, Ikezuki, 2002; Schönfelder,

Wittloht et al. 2002; Takeuchi and Tsutsumi 2002; Sugiura-Ogasawara, Ozaki et al.

2005; for review Vandenberg, 2007). Also, BPA has no tendency to accumulate; the

half life in the body is estimated to be less than one day (Pottenger, Domoradzki et

al. 2000; Takahashi and Oishi 2000). Therefore the contribution of HD to the overall

body burden is only marginal and the risk for the patient practically non-existent.

This is especially true if one compares the estimated additional body burden of

61.4 ng/kg bw to the tolerable daily intake of 50 µg/kg bw established by the

European Food Safety Agency (EFSA 2006). Of course it has to be considered that

the TDI was only established for oral exposure and not intravenous exposure. After

oral uptake, BPA is rapidly transformed to BPA glucoronide during the first pass

metabolism in the gut wall and the liver. The glucoronide form lacks endocrine

activity. If BPA leaches directly into the blood this metabolic biotransformation takes

longer and more BPA is available as parent BPA. However, bioavailability and activity

of parent BPA is reduced because it binds rapidly to human plasma proteins.

Even if this was not the case, there is still a safety factor of nearly 1000 between

the oral TDI and the actual intravenous exposure, which can be regarded as

sufficient to minimise the potential risk.

1.2 DEHP

Although DEHP could be detected in the eluates, the amounts were too low

reach the limit of quantification. This was surprising as we expected quantifiable

amounts of DEHP to leach from the blood tubing system. This expectation originated

from studies which report that patients undergoing maintenance hemodialysis retain

3.6 - 59.6 mg DEHP per dialysis session (average 16.4) (Faouzi, Dine et al. 1999;

Dine, Luyckx et al. 2000). An older study found even higher concentrations 23.8 -

360 mg leaching from the dialyser into the blood during a single session (average

E Discussion Toxicity of Eluates

117

105 mg) (Pollack, Buchanan et al. 1985). Given that leaching plasticizers – especially

leaching DEHP – have been recognized as a possible risk to human health, there

has been some effort to reduce the amount of leaching DEHP. Therefore values of

the more recent study will probably reflect the present situation more accurately.

Assuming the average amount of leaching DEHP would had been 16 mg, the

concentration in the 4 ml concentrated eluate would have been 4 mg/ml which is

considerably higher than the limit of quantification (ca. 0.07 µg/ml).

In order to explain this discrepancy, it has to be considered that the extraction

conditions of the present study differ from in vivo ones. In previous studies, the DEHP

was measured directly in the blood of HD patients. The complete amount of leaching

DEHP is stated as the difference between DEHP prior to a dialysis session and

thereafter. We on the other hand used ddH2O and 17.2 % EtOH for DEHP extraction.

As the extraction properties of blood and water are very different, it is possible that

our dialysis modalities failed to extract any DEHP. This seems plausible - at least in

regard to the ddH2O eluents - because DEHP solubility in water is very poor (41 µg/l

at 25°C (Leyder and Boulanger 1983)). However, this does not explain the complete

absence of DEHP in the ethanol eluates because DEHP migrates from PVC/DEHP

blends into ethanol/water mixtures (Kim, Kim et al. 2003). Apart from the different

eluent we also used only 1.1 m of tubing instead of several meters in the real HD,

which did lead to reduced amounts of potentially leaching DEHP. Finally, our

analytical method required the solution of DEHP in organic solvents, in our case

acetonitrile. During this process the eluents were diluted 1:10. All these factors may

have contributed to lower the concentration of DEHP. However, it can be concluded

that less DEHP was leaching from the tubing system used in this study than from

tubing systems of previous reports.

2 Toxicity of Eluates

Even though BPA was detected in each eluate, extensive toxicity testing yielded

no evidence for cyto- or genotoxicity. This is reassuring because the eluate

concentration reached in cell culture media was 25 times (EtOH eluates) to 50 times

(ddH2O eluates) higher than the expected concentration in the blood of HD patients.

This means there is an additional margin of safety.

Especially comforting is the lack of disturbed mitosis or reduced mitotic frequency

as well as the lack of MN in L5178Y cells exposed to eluates, given that BPA is

E Discussion Uremic Toxins

118

known to be a meiotic aneugen in female mice (Hunt, Koehler et al. 2003; Susiarjo,

Hassold et al. 2007). Other toxic effects of BPA include aneuploidy, disturbances in

the microtubule assembly, MN formation and DNA damage detectable by comet

assay in vitro, albeit at > 100 µg/ml while the BPA concentration reached in our test

system did not exceed the low ng range (Pfeiffer, Rosenberg et al. 1997; Tsutsui,

Tamura et al. 1998; Lee, Kwon et al. 2003). Therefore an induction of genomic

damage due to substances leaching from blood circuits ca not be expected.

However, EtOH eluates induced a slight increase in cell proliferation of the

estrogen sensitive cell line MCF-7. This increase of cell proliferation cannot solely be

attributed to BPA. A comparison to growth induction of pure BPA showed that

7.2 ng/ml BPA are necessary to increase the proliferation of MCF-7 cells significantly,

while the highest BPA concentration due to eluates was 5.8 ng/ml media (FX80).

Even EtOH eluates containing only a small amount of BPA (0.03 ng/ml media)

increased the cell proliferation. It can therefore be assumed that substances other

than BPA exhibited this estrogenic activity, or that BPA and another substance acted

synergistically.

We did not analyse for further substances but other groups report that repeated

flexion and compression of the tubing segment of HD by rollers of the peristaltic

pumps leads to abrasion of particles into the extracorporal circuit (Barron, Harbottle

et al. 1986).

Regardless of which substance of the eluate exhibits this estrogenic activity, the

relevance for the enhanced cancer incidence is limited. If the increased cancer

incidence was induced by estrogenic activity, one would assume that cancers of

estrogen responsive tissue (e.g. breast cancer) were the most prominent. However,

this is not the case in epidemiological studies; the incidence of breast cancer

incidences does not differ from the general population in a statistically significant way

(Maisonneuve, Agodoa et al. 1999; Teschner, Garte et al. 2002; Stewart, Buccianti et

al. 2003).

3 Uremic Toxins

3.1 Homocysteine

Low millimolar levels of Hcy induced MN in several cell lines. Similarly,

preliminary in vitro studies on pooled human lymphocytes showed an increased MN


119

frequency after incubation with Hcy (Crott and Fenech 2001). This is in line with in

vivo observations which correlate increased levels of Hcy in serum with an increase

in MN frequency (Fenech, Dreosti et al. 1997; Fenech, Aitken et al. 1998; Fenech

1999). So far the mechanism by which Hcy induces MN is not known. One of our

hypotheses was that MN induction is connected to disturbances of the cell cycle.

Disturbance of the cell cycle progression, i.e. an increased percentage of cells in the

S-phase were observed at the same concentration as MN. A prolonged S-phase can

result from changes in the level of DNA-cytosine-methylation. This is interesting in

the present context, since the DNA of dialysis patients, suffering from

homocysteinemia is often hypomethylated (Ingrosso, Cimmino et al. 2003) or shows

aberrant methylation patterns (Zaina, Lindholm et al. 2005). Increased plasma levels

have also been associated with increased DNA methylation of lymphocytes in healthy

subjects (Yi, Melnyk et al. 2000; James, Melnyk et al. 2002). The hypothesis for the

mechanism leading to hypomethylation is as follows:

The conversion of SAH to Hcy is a readily reversible reaction, which strongly

favours SAH synthesis instead of hydrolysis. The reason for the normal hydrolysis

reaction is the fast product removal (Yi, Melnyk et al. 2000). If the Hcy concentration

increases, the SAH concentration increases as well (Hoffman, Marion et al. 1980).

SAH in turn is a potent inhibitor of the SAM methyltransferase (Hoffman, Marion et al.

1980). If SAM-methyltransferases are inhibited, the methyltransferation of SAM to

DNA and other methyl acceptors drops. This leads to DNA hypomethylation.

However, neither Hcy nor Hcy-T treatment up to cytotoxic concentrations caused

an overall DNA-cytosine-methylation change in vitro. This is in contrast to the

situation in ESRD patients, in which hyperhomocystemia is correlated with

hypomethylation (Ingrosso, Cimmino et al. 2003). One possible explanation could be

a fast removal of Hcy by the transsulfuration pathway in the cell lines used within this

study. This would prevent the accumulation of SAH, the inhibitor of the SAM

methyltransferase.

Another possible mechanism for genotoxicity is the generation of ROS. Indeed, it

has been proposed that some of the adverse effects associated with

hyperhomocysteinemia may be due to the generation of ROS (Au-Yeung, Woo et al.

2004; Perez-de-Arce, Foncea et al. 2005). However, prior treatment of L5178Y cells

or co-incubation with oxygen scavengers like NAC did not reduce the MN frequency.

The comet assays of Hcy-treated cells yielded no evidence for oxidative DNA


120

damage. Hcy treatment had also no impact on the amount of ROS in HL60 cells, as

detectable by DCFH-DA analysis. This is in line with prior observations in HUVECS

(Outinen, Sood et al. 1998) and LLC-PK1 cells (Nakanishi, Akabane et al. 2005), in

which Hcy failed to elicit an oxidative stress response. Thus the generation of

oxidative stress by Hcy may be cell-type specific and /or dependent on experimental

conditions. This idea is supported by publications stating that Hcy (50 µM) increases

the ROS production of bovine aortic endothelial cells (BAEC) under high glucose but

not under normal growth condition (Sethi, Lees et al. 2006). It can inhibit as well as

promote LDL oxidation depending on the experimental conditions (Lynch, Campione

et al. 2000).

Interestingly, pre-treatment with Hcy caused even a considerable reduction of

oxidative stress in cells challenged by H2O2. This effect is probably due to a

conversion of Hcy to the major redox buffer of cells: GSH. This conversion takes

place in the transsulfuration reaction which provides the direct link between Hcy and

GSH (Mosharov, Cranford et al. 2000). In case of oxidative stress, the Hcy flux

through the transsulfuration pathway in liver cells is 2-3 times enhanced (Mosharov,

Cranford et al. 2000). This precedence over the transmethylation pathway is

controlled by the methionine synthase, whose activity is decreased under oxidizing

conditions (Chen, Pettersson et al. 1998), and the cystationine β-synthase, whose

activity increases under oxidizing conditions (Taoka, Ohja et al. 1998).

In fact, the GSH levels of HL60 and L5178Y cells started to increase as early as

30 min after incubation with Hcy. A similar increase of total intracellular GSH could be

observed in BAEC after incubation with 5 mM Hcy (Upchurch, Welch et al. 1997) or

50 µM Hcy (Sethi, Lees et al. 2006) or in DAMI cells (human megakaryocytic cells)

after incubation with 1 mM – 10 mM Hcy (Austin, Sood et al. 1998). However,

Upchurch et al. report that this was accompanied by a decreased glutathione

peroxidase activity.

After these experiments, ROS production or disturbances of the DNA methylation

can be ruled out as mechanisms for MN induction in L5178Y or HL60 cells. One

remaining explanation may be the prolonged S-phase after incubation with Hcy. This

may possibly lead to disturbances of the mitosis, which may lead to MN formation.

However, MN induction in L5178Y cells started after addition of 3 mM Hcy, while the

first significant cytotoxicity was observed at 4 mM Hcy. This slight difference could be

explained by the possibility that Hcy does not act directly genotoxic but non genotoxic


121

mechanisms are involved. The increased MN frequency of HL60 cells cannot be

explained in this way because no cytotoxicity was observed in this concentration

range.

3.1.1 Consequences for the Patient

Hcy exhibits genotoxic or antioxidant effects at concentrations significantly higher

than the levels observed in the serum of ESRD patients (up to 220 µM; (Perna, Satta

et al. 2006)). Although this may lead to the conclusion that these effects are

negligible for the patients, one can not rule out that – upon local accumulation in

certain tissues or cell types – toxicologically relevant levels may be reached.

Additionally, other more sensitive endpoints - e.g. gene expression measurements –

might detect Hcy-induced alterations at lower doses.

One particular interesting hypothesis can be put forward in regard to the

conversion of Hcy to GSH. In HD patients additionally suffering from chronic

malnutrition inflammation complex a high rather than a low Hcy level is correlated to

longer survival (Kalantar-Zadeh, Block et al. 2004; Wrone, Hornberger et al. 2004;

Ducloux, Klein et al. 2006). In those patients, the GSH level drops due to a disturbed

thiol homeostasis (Wlodek, Smolenski et al. 2006). Considering this, a conversion of

Hcy to GSH could enhance the oxidant defence, which contributes to the better

survival. The same mechanism has been observed in malnourished children – their

chances for survival are better when given GSH (Becker, Pons-Kuhnemann et al.

2005).

3.2 Homocysteine-Thiolactone

The cytotoxic and genotoxic effects of the Hcy derivate Hcy-T were also

analysed. Hcy-T was more cytotoxic towards L5178Y cells than Hcy. A decline of cell

proliferation could be observed at 1 mM Hcy-T. The MN frequency increased at the

same time. This is in line with studies of HL60 cells, in which severe cell death

occurred at concentrations of 500 – 1000 µM (Huang, Huang et al. 2002). Our

experiments with Hcy-T were troubled by the short half-life of 1 h and the

purchasable form which was only as hydrochloride. This resulted in an acidification of

the cell culture media which is problematic. Furthermore, the concentrations of Hcy-T

in plasma are even 3 powers lower than the ones of total Hcy (Chwatko and

Jakubowski 2005) its relevance for the in vivo situation is therefore highly unlikely.


122

3.3 Advanced Glycation End-Products

The two AGEs tested (MGO-BSA and CML-BSA) induced some genotoxicity

detectable by comet assay. However, the degree of genomic damage varied highly

between the batches of synthesized AGEs. Additionally, newly synthesized AGEs

lost their genotoxic capacity within days even if stored at -80°C. Some of these

problems have been overcome in another study and the genotoxic activity of AGEs

on LLC-PK1 and additional cell lines has been confirmed (Schupp, Schinzel et al.

2005). However, the concentrations reached in cell culture are significantly higher

(1 mg/ml versus 110 ng/ml) than in the HD patients. Nevertheless, AGEs are a

heterogeneous group of proteins; therefore it is possible that other AGEs are even

more genotoxic than the ones tested. The mechanism of genotoxicity was not

analysed in this study, however it is hypothesised that AGEs act via the RAGE

receptor. Activation of RAGE leads to activation of NF-κB, which leads to increased

cytokine, chemokine growth factor and ROS production (Sebekova, Wagner et al.

2007). Finally, this leads to oxidative DNA damage.

3.4 Leptin

Leptin did not induce any cytotoxicity at concentrations of up to 10 µg/ml, while it

did induce some genomic damage detectable by comet assay starting at 1 µg/ml.

This is roughly 10 times as much as the average leptin concentration in HD patients

(Vanholder, De Smet et al. 2003). The maximum amount of leptin detected in uremic

patients is 0.49 µg/l (Vanholder, De Smet et al. 2003). Therefore it is possible that

leptin contributes to the genomic damage observed in HD patients. This is supported

by the observation of leptin levels correlating with the peripheral genomic damage of

HD patients (Horoz, Bolukbas et al. 2006).

3.5 Serum of Dialysis Patients

None of the uremic toxins tested within this study provides a sufficient

explanation for the increased genomic damage observed in ESRD patients.

In order to evaluate whether uremic toxins are responsible for this problem or

play a minor part, L5178Y cells were incubated with serum or 10 kDa filtrate of HD

patients with increased MN frequency. Addition of serum or 10 kDa filtrate did not

induce cyto- or genotoxicity up to a concentration of 2% or 20%, respectively.

However, higher concentrations were severely cytotoxic. This severe cytotoxicity

E Discussion Conclusion

123

cannot be attributed to the uremic toxins alone. It is very likely that this cytotoxicity

was due to inadequate inactivation of the complement system.

The non-cytotoxic concentration of 2% serum in media is considerably lower than

the concentration to which lymphocytes are exposed. Therefore the results of tests

with patient serum or 10 kDa filtrate do not allow any conclusion as to whether

substances present in the serum are responsible for the genomic damage observed

in dialysis patients.

4 Conclusion

Based on the in vitro tests with eluates and the chemical analysis of eluates, it

can be concluded that substances leaching from dialysers pose no risk for the HD

patients - at least regarding the toxicity endpoints analysed.

It is therefore unlikely that substances leaching from dialysers are responsible for

the genomic damage and increased cancer incidence. This holds especially true as

most of the increased cancer incidence is observed within the first year after the start

of dialysis (Stewart, Buccianti et al. 2003). Cancer is a slowly progressing disease

which takes years to decades to develop. The rapid diagnosis after the start of HD,

supports the argument for detection because of better surveillance, not for cancer

development due to substances leaching from dialysers.

It is therefore more likely that uremic toxins play a role as they start to

accumulate as renal filtration decreases. Apart form the uremic toxins discussed

above, several additional uremic toxins have been tested for genotoxicity (indole-3-

acetic acid, Indoxyl sulphat and methylguanidine). All of them were genotoxic in vitro,

albeit at (much) higher concentrations than the ones reached in HD patients (Werner,

2005). This means that neither of those uremic toxins is sufficient to explain the

increased genomic damage or cancer incidence observed in dialysis patients.

Therefore it is reasonable to assume that the increased cancer incidence of HD

patients is a multifactorial problem. Other factors which certainly contribute to the

problem are:

1. Increased ROS production due to bio-incompability of dialysis membranes

2. an impaired DNA repair mechanism

3. an impaired antioxidant system

4. an impaired immune system

E Discussion Conclusion

124

Still an effect of uremic toxins cannot be dismissed easily. Even though the

concentrations in patients are generally lower than the concentrations exhibiting

genotoxicity in vitro, chronic exposure, the special susceptible of certain organs/cells

and synergistic effects of the various uremic toxins may lead to genotoxicity at lower

concentrations. Uremic toxins may also contribute to the DNA damage indirectly. The

accumulation of uremic toxins may impair the immune system, or disturb DNA repair.

It should therefore be examined whether patients may profit from an earlier onset of

dialysis or whether the disadvantages may outweigh the profit.

Overall, contributions of uremic toxins to the overall genomic damage seem

likely, as the reduction of uremic toxins by the more effective daily dialysis (compared

to the standard HD three times a week) was found to reduce the genomic damage

observed in dialysis patients.

F Zusammenfassung

125

F Zusammenfassung

Patienten, die an terminaler Niereninsuffizienz leiden und mittels Hämodialyse

behandelt werden, weisen einen erhöhten Genomschaden auf. Dieser könnte

ursächlich für die erhöhte Krebsinzidenz dieser Patientengruppe sein.

Eine der möglichen Ursachen für den erhöhten Genomschaden stellt die

Akkumulation genotoxischer Substanzen im Blut der Patienten dar. Diese

Substanzen können prinzipiell aus zwei unterschiedlichen Quellen stammen. Erstens

besteht die Möglichkeit, dass während der Dialyse Substanzen aus den Dialysatoren,

dem Blutschlauchsystem oder gar aus verunreinigtem Dialysat in das Blut der

Patienten übertreten. Zweitens führt der Verlust der Nierenfunktion zu einer stark

verminderten Exkretion harnpflichtiger Substanzen. Diese Substanzen akkumulieren

im Blut und bilden, sofern sie ein toxisches Potential besitzen, die Gruppe der so

genannten urämischen Toxine. Einige dieser urämischen Toxine sind potentiell auch

genotoxisch.

Im Rahmen der vorliegenden Dissertation wurden exemplarische Vertreter der

urämischen Toxine auf ihre genotoxische Wirkung hin untersucht. Außerdem wurde

analysiert, ob Substanzen aus Dialysatormembranen oder dem Blutschlauchsystem

austreten und in in vitro-Toxizitätstests Effekte zeigen. Der Fokus der Analytik lag

hierbei auf dem Nachweis von Bisphenol A, dem Hauptbestandteil verschiedener

Kunststoffe die für Dialysatoren und Dialysatormembranen verwendet werden, sowie

auf Diethylhexylphthalat (DEHP), welches als Weichmacher vielen

Blutschlauchsystemen beigesetzt wird.

Hierfür wurde der Dialysevorgang mit Hilfe von 5 verschiedenen Dialysatortypen

und PVC-Blutschläuchen imitiert. Als Extraktionsmittelmittel wurde doppelt

destilliertes Wasser verwendet bzw. eine 17.2%-ige Ethanol/Wasser-Mischung, die

gegenüber BPA ein ähnliches Extraktionsvermögen besitzt wie Blut. Die Temperatur,

Dialysedauer und Fließgeschwindigkeit entsprach weitgehend realistischen

Dialysemodalitäten. Auf diese Weise gewonnene Eluate wurden mittels HPLC-

MS/MS Analysen auf ihren Bisphenol A sowie Diethylhexylphthalat-Gehalt

untersucht. Während der DEHP-Gehalt in keinem der Eluate das

Quantifizierungslimit überschritt, wurden in allen Eluate zumindest Spuren von BPA

nachgewiesen.

F Zusammenfassung

126

Aus diesen Messungen ließ sich abschätzen, dass je nach Dialysedauer,

Dialysatortyp und Extraktionsmittel zwischen 6,4 ng und 4,3 µg Bisphenol A pro

Dialysator austraten. Die Menge an nachgewiesenem Bisphenol A war dabei in den

mit 17.2% Ethanol gewonnenen Eluaten deutlich höher als in den Wasser-Eluaten.

Des Weiteren zeigten sich deutliche Unterschiede zwischen den einzelnen

Dialysatortypen und Dialysatorchargen. Legt man für die Berechnung der

zusätzlichen Bisphenol A-Belastung eines Dialysepatienten den ungünstigsten Fall,

d.h. die höchste gemessenen BPA-Konzentration zugrunde, so erhält man einen

Wert von 0,84 ng/ml Blut (5 l) oder 61,4 ng/kg Körpergewicht (70 kg Person) pro

Dialyse.

Um das daraus resultierende Gefahrenpotential für den Dialysepatienten

abzuschätzen, mussten diese Werte mit den bisher in der Normalbevölkerung

gemessenen BPA-Plasmaspiegeln verglichen werden. Diese lagen zwischen 0,3 und

18,9 ng/ml Blut. Die zusätzliche Belastung durch die Dialyse liegt also eher im

unteren Bereich der Belastung der Normalbevölkerung. Außerdem besitzt Bisphenol

A eine kurze Halbwertszeit (> 1 Tag) und weist keine Tendenz zur Bioakkumulation

auf. Die zusätzlich Belastung von im schlimmsten Fall 61,4 ng/kg Körpergewicht liegt

außerdem noch mehr als den Faktor von 800 unter den derzeitig gültigen

Grenzwerten der Europäischen Behörde für Lebensmittelsicherheit (EFSA) für die

täglich tolerierbare orale Aufnahme (50 µg/kg Körpergewicht).

Da jedoch weitere - im Rahmen dieser Arbeit nicht näher identifizierte –

Substanzen in den Eluaten vorhanden waren, konnte eine Gefährdung der Patienten

zunächst nicht gänzlich ausgeschlossen werden. Daher wurden zusätzlich in vitro

Zyto- und Genotoxizitätstests durchgeführt. Die Konzentration der Eluate im

Zellkulturmedium war dabei 25 (Ethanoleluate) bis 50 (Wassereluate) mal höher als

die Konzentration, welche im Blut erreicht werden könnten. In keinem dieser Tests

ließ sich ein Hinweis auf eine zytotoxische oder genotoxische Wirkung finden. Es

konnte lediglich eine leichte östrogene Aktivität der mit Ethanol gewonnenen Eluate

nachgewiesen werden. Da BPA tatsächlich östrogene Kapazität besitzt, wurden

vergleichenden Versuchen mit reinem BPA durchgeführt. In diesen konnte erst ab

7,2 ng/ml ein signifikanter östrogener Effekt nachgewiesen werden. Da die BPA

Konzentration durch Eluate im Medium nur zwischen 0,03 ng/ml und 5,8 ng/ml lag

konnte dieser Effekt nicht ausschließlich auf austretendes BPA zurückzuführen sein.

Trotz dieser leichten östogenen Aktivität der Eluate kann das Gefahrenpotential für

F Zusammenfassung

127

den Dialysepatienten als minimal eingeschätzt werden. Xenoöstrogene können zwar

durchaus bei der Krebsentstehung eine Rolle spielen, allerdings wäre dann eine

erhöhte Tumorinzidenz von hormonresponsiven Geweben, wie z.B. Brustgewebe, zu

erwarten. Dies ist bei Dialysepatienten jedoch nicht der Fall.

Der zweite Teil der Arbeit fokussierte auf genotoxische Untersuchungen

exemplarischer urämischer Toxine. Da im Moment mehr als 90 verschiedene

urämische Toxine bekannt sind, musste zunächst eine Auswahl potentiell

genotoxischer Kandidaten getroffen werden. Der Schwerpunkt lag dabei auf

Homocystein, einem Zwischenprodukt des Methioninkreislaufs. Ein erhöhter

Homocysteinspiegel im Plasma von gesunden Menschen korreliert mit einer

erhöhten Mikrokernfrequenz. Auch in den im Rahmen dieser Studien durchgeführten

in vitro Versuchen führte die Exposition verschiedener Zelllinien mit Homocystein zu

einer erhöhten Mikrokernfrequenz. Allerdings erhöhte erst eine Inkubation mit 3 mM

Homocystein die Mikrokernfrequenz in diversen Zelllinien signifikant, während der

Homocysteinspiegel von Dialysepatienten nur in sehr schweren Fällen über 100 µM

ansteigt. Im Kometen-Test (einem weitern Genotoxizitätstest) ließ sich jedoch bis zu

zytotoxischen Konzentrationen kein erhöhter Genomschadennachweisen.

Mikrokerne entstehen häufig durch Störungen des Spindelapparates. Dies macht

sich u. U. in einer Störung des Zellzyklus bemerkbar. Jedoch übte Homocystein erst

ab einer Konzentration von 5 mM Homocystein einen statistisch signifikanten Einfluss

auf den Zellzyklus aus. Es erhöhte den prozentualen Anteil von Zellen in der S-

Phase. Da ein erhöhter Homocysteinspiegel bei Dialysepatienten auch mit einer

verminderten DNA-Cytosin-Methylierung korrelierte, wurde überprüft, ob dies für die

Verlängerung der S-Phase verantwortlich sein könnte. Konzentrationen bis zu 5 mM

änderten die DNA-Cytosin-Methylierung jedoch nicht. Höhere Konzentrationen

wurden nicht untersucht, da sie sich als zytotoxisch erwiesen.

Eine weitere Ursache für Genomschaden ist häufig die Entstehung von freien

Radikalen in der Zelle. Zugaben des Radikalfängers N-Acetylcystein verminderte

jedoch nicht die Mikrokernentstehung. Auch die Messung reaktiver Sauerstoffspezies

mittels Durchflusscytometrie erbrachten keinen Hinweis auf oxidativen Stress. Im

Gegenteil, durch Wasserstoffperoxyd ausgelöster Radikalstress wurde durch Co-

Inkubation mit Homocystein deutlich reduziert. Dies lag vermutlich an der

Umwandlung von Homocystein zu dem intrazellulären Antioxidanz Gluthathion.

F Zusammenfassung

128

Radikalstress konnte als Ursache für die Mikrokernentstehung also ausgeschlossen

werden.

Des Weiteren wurden Mikrokerntests mit dem Homocystein Derivat

Homocystein-Thiolacton durchgeführt. Wenn Homocystein bei der

Proteinbiosynthese irrtümlicherweise an Stelle von Methionin aktiviert wird, wandelt

es die methionyl-tRNA in Homocystein-Thiolacton um. Daher steigt mit einem

erhöhten Homocysteinspiegel auch der Homocysteine-Thiolactonspiegel in der Zelle.

Inkubation mit 1 mM Homocystein-Thiolacton erhöhte die Mikrokernfrequenz in vitro

signifikant. Allerdings zeigte sich gleichzeitig eine deutliche Zytotoxizität, wodurch ein

direkter genotoxischer Mechanismus nicht eindeutig nachgewiesen werden konnte.

Weitere interessante urämische Toxine sind die so genannten "Advanced

Glycation End-Products" (AGEs). Sie entstehen durch die nicht-enzymatische

Reaktion von reduzierenden Zuckern mit freien Aminogruppen von Peptiden oder

Proteinen. AGEs sind also eine sehr heterogene Gruppe von Proteinen. Zwei

exemplarische Vertreter wurden synthetisiert und im Komten-Test eingesetzt. Dabei

zeigte sich eine gewisse Genotoxizität, die allerdings sehr chargenabhängig war.

Als letztes wurde die toxische Wirkung des potentiellen urämischen Toxins Leptin

untersucht. Leptin ist ein Hormon, welches hauptsächlich von Adipocyten hergestellt

wird und für die Regulation der Nahrungsaufnahme und des Energieverbrauchs

zuständig ist. In der Zellkultur induzierte Leptin einen ab einer Konzentration von

1µg/ml Genomschäden, die im Kometen-Test nachweisbar waren. Es induzierte

jedoch keine Mikrokerne oder zeigte Zytotoxizität.

Zusammenfassend lässt sich somit sagen, dass mehrere urämische Toxine in

vitro eine genotoxische Wirkung entfalten, allerdings erst in Konzentrationen die in

Patienten nicht erreicht werden. Natürlich lässt sich nicht ausschließen, dass diese

durch die chronische Exposition in sensitiven Geweben oder Zelltypen schon bei

physiologisch relevanten Konzentrationen auftreten könnten. Wahrscheinlicher ist

jedoch, dass das Problem der erhöhten Genomschäden multifaktoriell ist. Neben der

eventuell sogar synergistischen Wirkung der urämischen Toxine, spielen vermutlich

auch noch folgende Faktoren eine Rolle: (1.) vermehrte Sauerstoffradikalenbildung

durch Inkompatibilität zwischen Dialysemembranen und Blut, (2.) verschlechterte

DNA-Reparatur der Dialysepatienten (3.) ein geschwächtes Immunsystem sowie (4.)

ein geschwächtes Antioxidanzsystem.

G Summary

129

G Summary

In patients suffering from end-stage renal disease who are treated by

hemodialysis genomic damage as well as cancer incidence is elevated.

One possible cause for the increased genomic damage could be the

accumulation of genotoxic substances in the blood of patients. Two possible sources

for those toxins have to be considered. The first possibility is that substances from

dialysers, the blood tubing system or even contaminated dialysis solutions may leach

into the blood of the patients during dialysis. Secondly, the loss of renal filtration

leads to an accumulation of substances which are normally excreted by the kidney. If

those substances possess toxic potential, they are called uremic toxins. Several of

these uremic toxins are potentially genotoxic.

Within this thesis several exemplary uremic toxins have been tested for genotoxic

effects. Additionally, it was analysed whether substances are leaching from dialysers

or blood tubing and whether they cause effects in in vitro toxicity testing. The focus of

chemical analytisis was on bisphenol A (BPA), the main component of plastics used

in dialysers and dialyser membranes, as well as on di(ethylhexyl)phthalate (DEHP),

which is used as plasticiser in many blood tubing systems.

For this purpose dialysis was simulated using five different kinds of dialysers and

PVC blood tubing. Two different eluents (reverse osmotic water and 17.2% ethanol)

were used while temperature, dialysis period and flow rate conformed to the real

dialysis modalities. The eluates obtained were analysed by LC-MS/MS for their BPA

as well as DEHP content. The DEHP concentration did not exceed the limit of

quantification in any eluate. In contrast, all of the eluates contained quantifiable

amounts of BPA.

From these results it was extrapolated that 6.4 ng to 4.3 µg BPA are leaching per

dialyser. The amount of BPA depended on the duration of dialysis, the type of

dialyser and the eluent. The amount of leaching BPA was higher when 17.2%

ethanol was used. This is an eluent which has similar extraction properties as blood.

Additionally substantial differences between different batches of dialysers could be

detected. Assuming the worst case –we estimated the additional body burden of BPA

to be 0.84 ng/ml blood (5 l blood) or 61.4 ng/ kg body weight (70 kg person) per

dialysis session.

G Summary

130

In order to estimate the potential risk for the patient, these values had to be

compared to the plasma levels of average humans. Those values are between 0.3 –

18.9 ng/ml blood. One also has to take into consideration that BPA has no tendency

for bioaccumulation and a short half-life (< 1 day). Therefore the additional burden

due to dialysis is in the low range of the average body burden. Furthermore, the

additional body burden of 61.4 ng/ kg body weight is still a factor of more than 800

below the threshold of the European Food Safety Agency (EFSA) for the tolerable

daily oral intake (50 µg/kg bw).

However, the eluates contained additional substances which have not been

identified in this study. For this reason a potential risk for the patient could not initially

be ruled out. As a consequence in vitro cytotoxicity and genotoxicity tests were

conducted. The concentration of eluates in cell culture media was 25 (ethanol

eluates) to 50 times (water eluates) higher than the one reached in blood. No

evidence for cytotoxicity or genotoxicity could be found in any of the tests. Solely the

test for estrogenic activity yielded slightly positive results when performed with the

ethanol eluates. As BPA possesses estrogenic activity, comparative experiments with

pure BPA were conducted. BPA showed a statistically significant estrogenic effect at

7.2 ng/ml, while the BPA concentration in the cell culture media was between 0.03

ng/ml and 5.8 ng/ml. The estrogenic activity of the eluates could therefore not have

been caused by BPA exclusively. Despite the slight estrogenic activity, the risk for the

hemodialysis patient by leaching substances can be regarded as negligible. Although

it is known that xenoestrogens may contribute to tumour development, an increased

cancer incidence of hormone responsive tissue - like breast tissue - would be

expected. In dialysis patients, this is not the case.

The second part of the work focused on genotoxic testing of exemplary uremic

toxins. At the moment more than 90 different toxins are known, therefore the first task

was to choose potentially genotoxic ones. Most of the work focused on

homocysteine, an intermediate of the methionine cycle. Elevated plasma levels of

homocysteine have been correlated to an increased micronucleus frequency in

healthy persons. Micronuclei are markers for genomic damage. This correlation was

confirmed by in vitro tests with several cell lines during this study. Exposure to

homocysteine resulted in an increased micronucleus frequency. However, a

homocysteine concentration of 3 mM was necessary to induce micronuclei in vitro,

G Summary

131

while the plasma concentration of homocysteine in humans exceeds 100 µM only in

very severe cases. In the comet assay (a second genotoxicity test) no genomic

damage could be observed up to cytotoxic concentrations.

Frequently, micronuclei result from disturbances of the spindle apparatus. These

often lead to disturbances in the cell cycle. In fact, incubation with homocysteine did

increase the percentage of cells in S-phase. However, this was not the case until a

concentration of 5 mM homocysteine was reached in the cell culture medium. An

increased plasma level of homocysteine has also been correlated to a decreased

overall DNA cytosine methylation. Therefore we analysed whether changes of DNA

cytosine methylation are responsible for the extension of the S-phase. However, Hcy

concentrations of up to 5 mM did not change levels of DNA methylation. Higher

concentrations were not evaluated because they were cytotoxic.

Another common reason for increased genomic damage is the formation of free

radicals. However, addition of the radical scavenger N-acetylcysteine did not reduce

the micronucleus frequency. Flow cytometric measurements of reactive oxygen

species failed to detect oxidative stress due to Hcy. On the contrary, radical stress

induced by hydrogen peroxide could be reduced by co-incubation with homocysteine.

This was probably due to the intracellular conversion of homocysteine to the

intracellular antioxidant glutathione. Therefore oxidative stress can be ruled out as

cause for micronuclei.

Additionally, micronucleus tests with the derivate of homocysteine:

homocysteine-thiolactone have been performed. If homocysteine wrongly enters the

biosynthetic apparatus instead of methionine, it is activated and subsequently

converted to homocysteine-thiolactone. This explains the increasing level of

homocysteine-thiolactone if the homocysteine level in the cell increases. Incubation

of cells with 1 mM homocysteine-thiolactone increased the micronucleus frequency

significantly. However, this concentration was also cytotoxic which prevented the

unambiguous proof of direct genotoxicity.

Further uremic toxins of interest are the so-called advanced glycation end-

products. They result from non-enzymatic reaction of reducing sugars with free amino

groups of peptides and proteins and are a heterogenic group of proteins. Two

exemplary representatives of this group have been synthesized and tested in the

comet assay. They induced genotoxicity although the severity was depending on the

batch.

G Summary

132

Finally, the toxicity of the potential uremic toxin leptin was evaluated. Leptin is a

hormone which is mainly synthesized by adipocytes and which regulates food intake

and energy expenditure. Starting at 1 µg/ml leptin, induced genomic damage

detectable by comet assay. It did not induce micronuclei or cytotoxicity.

To summarize: several uremic toxins exhibit genotoxicity in vitro but only at

concentrations higher than those reached in the patient. Still it cannot be ruled out

that chronic exposure of sensitive tissue or cell types may have a genotoxic effect at

physiologically relevant concentrations. It is more likely that this problem is

multifactorial. Apart from possible synergistic effects of uremic toxins, other factors

are probably involved: (1.) increased formation of reactive oxygen species due to

incompatibility of dialysis membranes and blood, (2.) an impaired DNA repair system

of dialysis patients, (3.) a weakened immune system, or (4.) an reduced antioxidant

defence.

H Acknowledgements

133

H Acknowledgements

Foremost I would like to thank my supervisor Prof. Dr. Helga Stopper for her

active interest in my work, her consistently great support and her expert guidance.

I warmly thank Prof. Dr. Vienken and Prof. Dr. Heidland for their fruitful

discussion about my work and their excellent advice.

My thanks go also to Prof. Dr. Benz and Prof. Dr. Nieswand for supervising my

work on behalf of the Faculty of Biology and the International Graduate School of the

University of Würzburg.

I would like to thank the past and the present members of the Stopper lab for

providing a great work environment, sharing the ups and down of a grad student's life

and for their help and chats.

Special thanks to Judith Manz and Theresa Ehrlich for their assistance with the

laboratory work.

I also owe a lot of thanks to Andreas Brink, Eva Trösken, Nataly Bittner and

Wolfgang Völkel for their help with mass-spectrometry.

I am especially grateful to Cristina Bouchcinsky for reading my manuscript

This research has been founded by Fresenius medical care. I gratefully

acknowledge this support.

Finally, I want to thank Reinhard Fink for his loving support and his patience

during the last stages of my PhD.

I Appendix List of Abbreviations

134

I Appendix

1 List of Abbreviations

2-EH 2-Ethylhexanol

5-Aza-C 5-Aza-cytidine

5cx-MEPP Mono-[2-ethyl-5-carboxypentyl] phthalate

5cx-MMHP Mono-[2-(carboxymethyl)hexyl] phthalate

5-mdCyd 5-Methyl-2`deoxycytidine

5OH-MEHP Mono-[2-ethyl-5-hydroxylhexyl] phthalate

5oxo-MEHP Mono-[2-ethyl-5oxylhexyl] phthalate

7-AAD 7-Aminoactinomycin

8-OHdG 8-Hydroxy-2-deoxy-Guanosin

AARS Aminoacryl-tRNA

AGE Advanced glycation end-product

AH Adenosyl-homocysteinase

AMP Adenosine monophosphate

AMU Atomic mass unit

ATP Adenosine triphosphate

ATSDR Agency for Toxic Substances and Disease Registry

BHMT Betaine-homocysteine-S-methyltransferase

BMI Body mass index

BPA Bisphenol A

BrdU 5-Bromo-2-deoxyuridine

BSA Bovine serum albumine

BW Body weight

CA Comet assay

CBS Cystathione-β-synthase

CIMS Chronic inflammation malnutrition syndrom

CML Carboxy(methyl)lysine

cps Counts per second

CRF Chronic renal failure

Cyt B Cytochalasin B

DCF 2`7`-Dichlorofluorescein

DCFH-DA 2`7`-Dichlorofluorescein diacetat

ddH2O Double-distilled water, HPLC-grade water

DEHP Di-ethylhexyl-phthalate

dGuo 2`-Deoxyguanosine

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DOP Dioctyl phthtalate

DTNB 5`5`-Dithiobis-2-nitrobenzoic acid

ECB European Chemical Bureau

EDTA Ethylene-diamine-tetraacetic acid

EFSA European Food Safety Agency

ELISA Enzyme-linked ImmunoSorbent Assay


135

EPA European Protection Agency

ER Estrogen Receptor

ESRD End-stage renal disease

EtOH Ethanol

FACS Fluorescence-activated cell-sorting

FBS Fetal bovine serum

FIGE Field inversion gel electrophoresis

FITC Fluorescein isothiocyanate

FSC Forward scatter

FSH Follicle stimulating hormone

g Gram

g Gravitational constant

GC-MS Gass-chromatography-mass spectrometry

GFR Glomerular filtration rate

GSH Glutathione (reduced form)

GSSG Glutathione (oxidized form)

h Hours

HCl Hydrochloric acid

Hcy Homocysteine

Hcy-T Homocysteine-thiolactone

HD Hemodialysis

HDF Hemodiafiltration

HDL High-density lipoproteins

HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC High-performance liquid chromatography

HPLC-MS/MS HPLC linked to a tandem-mass spectrometer

HPRT Hypoxanthine-guanine phosphoriboxyl transferase

HUVEC Human umbilical vein endothelial cells

l Liter

LC-MS Liquid chromatography-mass spetrometry

LC-MS/MS Liquid chromatography-tandem-mass spectrometer

LD50 Leathal dose, 50%

LDL Low-density lipoprotein

LMP-Agarosis Low melting point agarosis

LOAEL Lowest observed adverse effect level

LOD Limit of detection

LOEL Lowest observed effect level

JAK Janus kinase

M Mole

m/z Mass-charge-ratio

mA Milli Ampere

MAT Methionine-adenosyltransferase

MEHP Mono(2-ethylhexyl)phthalate

MET Methionine

MGO Methylglyoxal

min Minutes

MMC Mitomycin C

MMS Methylmethane sulfonate


136

MN Micronuclei

mRNA Messenger ribonucleic acid

MS Mass spectrometry

MS/MS Tandem mass spectrometry

MW Molecular weight

µg Microgram

µl Microliter

µM Micromolar

n Normal

NAC N-acetylcysteine

NaCNBH3 Sodium cyanoborohydride

NADP+ Nicotinamide adenine dinucleotide phosphate (oxidized form)

NADPH Nicotinamide adenine dinucleotide phosphate (reduced form)

NaOH Sodium hydroxide

NF-κB Nuclear factor-kappa B

NOEL No observed effect level

p Probability

PAGE Polyacrylamide gel electrophoresis

PBS Phosphate-buffered saline

PBS-CMF Phosphate buffered saline – calcium and magnesium free

PD Peritoneal dialysis

PI Propidium iodide

Ppar-α Peroxisome proliferators-activated receptor α

P/S Penicillin / streptomycine

PS Horse serum

PVC Polyvinyl chloride

RAGE Receptor for advanced glycation end products

RNA Ribonucleic acid

ROS Reactive oxygen species

sec Seconds

S.D. Standard Deviation

SAH S-adenosylhomocysteine

SAM S-adenosylmethionine

SIR Standardized incidence ratio

SSC Side scatter

STAT Signal transducers and activators of transcription

TDI Tolerable daily intake

tHcy Total homocysteine

THF Tetrahydrofolate

TRIS Trishydroxymethylaminomethane

tRNA Transfer RNA

PVP Polyvinylpyrrolidon

I Appendix Figures

137

2 Figures

Fig. A 1 Kidney anatomy ..........................................................................................1

Fig. A 2 Schematic picture of a hemodialysis circuit ...............................................4

Fig. A 3 Molecular structure of bisphenol A..............................................................7

Fig. A 4 Molecular structure of di-(2-ethylhexyl)phthalate ......................................12

Fig. A 5 Metabolism of di(2-ethylhexyl)phthalate ...................................................14

Fig. A 6 The structure of homocysteine..................................................................20

Fig. A 7 Hcy metabolism and the enzymes and vitamins involved ........................22

Fig. A 8 Equation for the formation of Hcy-T .........................................................28

Fig. A 9 Hcy/Hcy-T metabolism .............................................................................28

Fig. A 10 Formation of AGEs ..................................................................................30

Fig. C 1 Exemplary dot plot of a quantitative cell cycle analysis ............................45

Fig. C 2 Exemplary pictures of cells used in the comet-assay ...............................46

Fig. C 3 Schematic formation of micronuclei ..........................................................47

Fig. C 4 Fate of cells following exposure to cytotoxic/genotoxic agents ................49

Fig. C 5 Exemplary histogram of a flow cytometric analysis of L5178Y cells, stained against 5’methyl-cytosine .............................................................54

Fig. C 6 Exemplary oxidative stress measurement of HL60 cells .........................59

Fig. C 7 Reactions of the GSH-GSSG assay .........................................................60

Fig. C 8 L5178Y cells stained with Hoechst 33342 ................................................62

Fig. C 9 Annexin-V/PI staining of L5178Y cells. .....................................................64

Fig. C 10 Assembly of the dialysis equipment..........................................................70

Fig. D 1 Lyophilised eluate in a 250 ml flask ..........................................................75

Fig. D 2 Chromatograms of full range scans .........................................................76

Fig. D 3 Exemplary mass spectra...........................................................................77

Fig. D 4 Chromatogram overlay of an Eluat and 100 pg/µl DEHP standard ..........78

Fig. D 5 Mass spectrum of 100 pg/µl DEHP standard............................................78

Fig. D 6 Chromatogram of an eluat .......................................................................79

Fig. D 7 Mass spectrum of bisphenol A..................................................................79

Fig. D 8 Relative cell proliferation of L5178Y cells after incubation with 4 h eluates for 24 h. ........................................................................................83

Fig. D 9 Relative cell proliferation of L5178Y cells after incubation with 24 h eluates for 24 h. .......................................................................................83

Fig. D 10 Relative mitosis frequency of L5178Y cells after incubation with 4 h eluates for 24 h. .......................................................................................84

I Appendix Figures

138

Fig. D 11 Relative mitosis frequency of L5178Y cells after incubation with 24 h eluates for 24 h. .......................................................................................84

Fig. D 12 Percentage of apoptotic L5178Y cells after incubation with 4 h and 24 h for 24 h. ............................................................................................85

Fig. D 13 Percentage of apoptotic L5178Y cells after incubation with 4 h eluates for 24 h. .......................................................................................86

Fig. D 14 Percentage of apoptotic L5178Y cells after incubation with 24 h eluates for 24 h. ........................................................................................86

Fig. D 15 Relative number of micronuclei per thousand L5178Y cells after incubation with 4 h eluates for 24 h. .........................................................87

Fig. D 16 Relative number of micronuclei per thousand L5178Y cells after incubation with 24 h for 24 h. ....................................................................88

Fig. D 17 Relative DNA damage of L5178Y cells detected by the comet assay after incubation with 4 h eluates for 24 h. ................................................89

Fig. D 18 Relative DNA damage of L5178Y cells detected by the comet assay after incubation with 24 h eluates for 24 h. ..............................................89

Fig. D 19 Relative proliferation of MCF-7 cells after 8 days incubation with 24 h eluates. ....................................................................................................90

Fig. D 20 Relative proliferation of MCF-7 cells after 8 days incubation with bisphenol a. .............................................................................................91

Fig. D 21 Proliferation of L5178Y cells after incubation with homocysteine for 24 h or 120 h.............................................................................................93

Fig. D 22 Proliferation relative to the control of L5178Y cells and HL60 cells after incubation with homocysteine ..........................................................94

Fig. D 23 Proliferation relative to the control of L5178Y cells after incubation with homocysteine-thiolactone .................................................................94

Fig. D 24 Percentage of cells undergoing mitosis in L5178Y cells and HL60 cells after incubation with homocysteine...................................................95

Fig. D 25 Percentage of cells undergoing apoptosis in L5178Y cells and HL60 cells after incubation with homocysteine for 24 h for 48 h.........................96

Fig. D 26 Percentage of cells undergoing apoptosis in L5178Y cells after incubation with homocysteine-thiolactone.................................................96

Fig. D 27 Micronucleus induction in L5178Y cells after incubation with homocysteine for 24 h or 120 h ................................................................97

Fig. D 28 Micronucleus induction in L5178Y cells and HL60 cells after incubation with homocysteine ...................................................................98

Fig. D 29 Micronucleus induction in L5178Y cells after incubation with homocysteine-thiolactone for 24 h. ...........................................................98

Fig. D 30 Micronucleus induction in Caco cells, TK6 cells and LLC-PK1 cells after incubation with homocysteine...........................................................99

Fig. D 31 DNA damage analysed by comet-assay after incubation of L5178Y cells and HL60 cells with homocysteine..................................................100

I Appendix Figures

139

Fig. D 32 DNA damage analysed by comet-assay after incubation of L5178Y cells with homocysteine-thiolactone for 24 h...........................................100

Fig. D 33 DNA damage analysed by comet-assay (after incubation of L5178Y cells 5 mM homocysteine for 2 - 24 h. ....................................................101

Fig. D 34 DNA damage analysed by comet-assay after incubation of L5178Y cells 5 mM homocysteine-thiolactone for 2 - 24 h...................................101

Fig. D 35 Micronucleus frequency of L5178Y cells after incubation with 3 mM homocysteine or homocysteine-thiolactone and N-acetylcysteine for 24 h.........................................................................................................102

Fig. D 36 Oxidative stress level measured after 30 to 120 minutes incubation of HL60 cells with 3 mM homocysteine or pretreated with homocysteine and challenged with H2O2 .......................................................................103

Fig. D 37 Oxidative stress level measured after 4 to 24 h incubation of HL60 cells with 3 mM homocysteine or pre-treated with homocysteine and challenged with H2O2 ..............................................................................103

Fig. D 38 Relative amount of GSH in L5178Y cells after incubation with 3 mM homocysteine for 0.5 to 24 h ..................................................................104

Fig. D 39 Relative amount of GSH in HL60 cells after incubation with 3 mM homocysteine for 0.5 to 24 h...................................................................104

Fig. D 40 Flow cytometric analysis of DNA-cytosine methylation in L5178Y cells after 72 h exposition of homocysteine. ...........................................105

Fig. D 41 Analysis of DNA-cytosine methylation in L5178Y cells after 72 h exposition of homocysteine by HPLC-MS/MS. .......................................106

Fig. D 42 Cell cycle analysis of L5178Y cells after 12 h exposure to homocysteine by BrdU incorporation assay............................................107

Fig. D 43 Cell cycle analysis of L5178Y cells after 12 h exposure to homocysteine by BrdU incorporation assay............................................107

Fig. D 44 Percentage of apoptotic L5178Y cells after 24 h incubation 0.1 – 10 µg/ml NaAsO2. ...................................................................................108

Fig. D 45 Micronucleus induction in L5178Y cells after incubation with leptin for 24 h.........................................................................................................109

Fig. D 46 DNA damage analysed by comet- after incubation of L5178Y cells with leptin for 24 h...................................................................................109

Fig. D 47 Exemplary comet-assay of LLC-PK1 cells incubated with AGEs for 24 h.........................................................................................................110

Fig. D 48 Proliferation after incubation of L5178Y cells after incubation of L5178Y cells with 2% patient serum for 1 week or with 20% 10 kDa filtrate of the patient serum for 24 h. .......................................................112

Fig. D 49 Micronucleus induction in L5178Y cells after incubation of L5178Y cells with 2% patient serum for 1 week or with 20% 10 kDa filtrate of the patient serum for 24 h. ......................................................................112

I Appendix Tables

140

3 Tables

Tab. A 1 Types of membranes with examples ..........................................................5

Tab. A 2 Site-specific cancer risk in ESRD patients ..................................................6

Tab. A 3 Median body burden of DEHP ..................................................................14

Tab. A 4 Examples of uremic toxins .......................................................................20

Tab. A 5 Determinants of plasma total Hcy .............................................................25

Tab. C 1 General technical equipment ....................................................................36

Tab. C 2 General materials and chemicals..............................................................37

Tab. C 3 Media and supplements............................................................................38

Tab. C 4 General buffer...........................................................................................38

Tab. C 5 Cell lines, media and growth conditions....................................................39

Tab. C 6 Frequent test substances..........................................................................42

Tab. C 7: Dialysers and their properties ....................................................................69

Tab. C 8 Utilized and obtained eluate from different dialysers ................................71

Tab. D 1 Elution modalities for DEHP measurement and results ............................78

Tab. D 2 Amount of BPA detected in different eluates. ...........................................80

Tab. D 3 Estimated amount of leaching BPA per dialyser .......................................81

Tab. D 4 Eluate concentrations in cell culture .........................................................82

Tab. D 5 Summary of the toxicity testing. ................................................................92

Tab. D 6 Summary of cytotoxic/genotoxic effects of uremic toxins on L5178Y or LLC-PK1 cells.....................................................................................111

I Appendix References

141

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I Appendix Curriculum vitae

154

5 Curriculum vitae

Kristin Fink (née Kobras)

Pestalozzistr. 7

74321 Bietigheim-Bissingen

* 7th May 1977, in Magdeburg, Germany

Current employment

01/2008 - present Project Manager "Toxicology", Dr-Knoell-Consult GmbH,

Mannheim

Education

01/2004 - present Graduate study and PhD thesis in toxicology at the

Department of Toxicology, University of Würzburg on:

"Toxins in renal disease and dialysis therapy: genotoxical

potential and mechanisms"

01/2005 - present Participant of the post-graduate education program

"Expert in Toxicology", DGPT

06/2004 - 12/2006 Associated member of the International Graduate College

"Target Proteins"

10/1997 - 07/2003 Undergraduate study of biology, University of Konstanz

Degree: Diploma in biology

08/2000 - 06/2001 Undergraduate study of biology, University of Guelph,

Canada

10/1996 - 09/1997 Undergraduate study of chemistry at the Albert-Ludwigs

University, Freiburg

08/1987 - 06/1996 Secondary school, Geschwister-Scholl-Gymnasium

Waldkirch

Degree: Abitur

08/1983 - 06/1987 Primary school, Glottertal

I Appendix Publications

155

6 Publications

Fink K, Brink A, Vienken J, Heidland A, Stopper H: Homocysteine exerts genotoxic

and antioxidative effects in vitro. Toxicol In Vitro. 2007 Dec; 21(8):1402-8.

Kobras K, Schupp N, Nehrlich K, Adelhardt M, Bahner U, Vienken J, Heidland A,

Sebekova K, Stopper H: Relation between different treatment modalities and

genomic damage of end-stage renal failure patients. Kidney Blood Press Res.

2006;29(1):10-7.

Schupp N, Stopper H, Rutkowski P, Kobras K, Nebel M, Bahner U, Vienken J,

Heidland A.: Effect of different hemodialysis regimens on genomic damage in end-

stage renal failure. Semin Nephrol. 2006 Jan;26(1):28-32. Review.

Brink A, Schulz B, Kobras K, Lutz WK, Stopper H.: Time-dependent effects of

sodium arsenite on DNA breakage and apoptosis observed in the comet assay.

Mutat Res. 2006 Feb 28;603(2):121-8.

Stopper H, Schmitt E, Kobras K: Genotoxicity of phytoestrogens. Mutat Res. 2005

Jul 1;574(1-2):139-55.

I Appendix Ehrenwörtliche Erklärung

156

7 Ehrenwörtliche Erklärung

Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und keine anderen als die

von mir angegebenen Quellen und Hilfsmittel benutzt habe.

Ferner erkläre ich, dass ich nicht versucht habe, diese Dissertation anderweitig mit

oder ohne Erfolg in gleicher oder ähnlicher Form einzureichen.

Ich habe keine Doktorprüfung an einer anderen Hochschule abgelegt oder endgültig

nicht bestanden.

Bietigheim-Bissingen, den

(Kristin Fink)

Documents

Toxins in Renal Disease and Dialysis Therapy: Genotoxic ... · dialysis patients is expected between 2000 and 2015 (Gilbertson, Liu et al. 2005), and 2 million ESRD patients world-wide